Methods for treating chronic kidney disease

ABSTRACT

The present invention relates to methods for treating chronic kidney disease (CKD) including methods for preventing or delaying onset of CKD and methods for preventing exacerbation and progression of CKD. In particular embodiments, the invention provides methods for treating a subject at risk of developing CKD comprising administering to the subject a composition comprising a) a therapeutically effective amount of at least one oligonucleotide compound which inhibits the expression of a human target gene associated with the kidney disease; and b) a pharmaceutically acceptable excipient or carrier, or mixtures thereof, thereby reducing the risk of CKD in the subject.

This application claims priority of U.S. Provisional Patent ApplicationNos. 61/184,937, filed 8 Jun. 2009 and 61/235,381, filed 20 Aug. 2009;both of which are hereby incorporated by reference in their entirety.

Throughout this application various patents and publications are cited.The disclosures of these documents in their entireties are herebyincorporated by reference into this application to more fully describethe state of the art to which this invention pertains.

FIELD OF THE INVENTION

The present invention relates to methods for treating chronic kidneydisease (CKD) including methods for preventing or delaying onset of CKDand methods for preventing exacerbation and progression of CKD. Inparticular embodiments, the invention provides methods for treating asubject at risk of developing CKD comprising administering to thesubject a composition comprising a) a therapeutically effective amountof at least one oligonucleotide compound which inhibits the expressionof a human target gene associated with the kidney disease; and b) apharmaceutically acceptable excipient or carrier, or mixtures thereof,thereby reducing the risk of CKD in the subject.

BACKGROUND OF THE INVENTION Chronic Kidney Disease

Chronic kidney disease (CKD) is a worldwide public health problem and isrecognized as a common condition that is associated with an increasedrisk of cardiovascular disease and end stage renal disease (ESRD).

The Kidney Disease Outcomes Quality Initiative (K/DOQI) of the NationalKidney Foundation (NKF) defines chronic kidney disease as either kidneydamage or a decreased kidney glomerular filtration rate (GFR) for threeor more months. Other markers of CKD are also known and used fordiagnosis. In general, the destruction of renal mass with irreversiblesclerosis and loss of nephrons leads to a progressive decline in GFR andeventually ESRD.

Recently, the K/DOQI published a classification of the stages of CKD, asfollows:

Stage 1: Kidney damage with normal or increased GFR (>90 mL/min/1.73 m²)Stage 2: Mild reduction in GFR (60-89 mL/min/1.73 m²)Stage 3: Moderate reduction in GFR (30-59 mL/min/1.73 m²)Stage 4: Severe reduction in GFR (15-29 mL/min/1.73 m²)Stage 5: Kidney failure (GFR<15 mL/min/1.73 m² or dialysis)In stages 1 and 2 CKD, GFR alone does not confirm the diagnosis. Othermarkers of kidney damage, including abnormalities in the composition ofblood or urine or abnormalities in imaging tests, should be relied upon.

Pathophysiology of CKD

Approximately 1 million nephrons are present in each kidney, eachcontributing to the total GFR. Irrespective of the etiology of renalinjury, with progressive destruction of nephrons, the kidney is able tomaintain GFR by hyperfiltration and compensatory hypertrophy of theremaining healthy nephrons. This nephron adaptability allows forcontinued normal clearance of plasma solutes so that substances such asurea and creatinine start to show significant increases in plasma levelsonly after total GFR has decreased to 50%, when the renal reserve hasbeen exhausted. The plasma creatinine value will approximately doublewith a 50% reduction in GFR. Therefore, a doubling in plasma creatininefrom a baseline value of 0.6 mg/dL to 1.2 mg/dL in a patient actuallyrepresents a loss of 50% of functioning nephron mass.

The residual nephron hyperfiltration and hypertrophy, althoughbeneficial for the reasons noted, is thought to represent a major causeof progressive renal dysfunction. This is believed to occur because ofincreased glomerular capillary pressure, which damages the capillariesand leads initially to focal and segmental glomerulosclerosis andeventually to global glomerulosclerosis. This hypothesis has been basedon studies of five-sixths nephrectomized rats, which develop lesionsthat are similar to those observed in humans with CKD.

The two most common causes of chronic kidney disease are diabetes andhypertension. Other factors include acute insults from nephrotoxins,including radiocontrast agents, or decreased perfusion (ischemia);sepsis; Proteinuria; Increased renal ammoniagenesis with interstitialinjury; Hyperlipidemia; Hyperphosphatemia with calcium phosphatedeposition; Decreased levels of nitrous oxide and smoking.

In the United States, the incidence and prevalence of CKD is rising,with poor outcomes and high cost to the health system. Kidney disease isthe ninth leading cause of death in the US. The high rate of mortalityhas led the US Surgeon General's mandate for America's citizenry,Healthy People 2010, to contain a chapter focused on CKD. The objectivesof this chapter are to articulate goals and to provide strategies toreduce the incidence, morbidity, mortality, and health costs of chronickidney disease in the United States. The burden of chronic kidneydisease can be assessed by multiple criteria, all of which underscorethe need for improved detection, treatment, and monitoring of clinicaland fiscal outcomes. Reducing renal failure will require additionalpublic health efforts, including effective preventive strategies andearly detection and treatment of chronic kidney disease.

The incidence rates of end-stage renal disease (ESRD) have alsoincreased steadily internationally since 1989. The United States has thehighest incident rate of ESRD, followed by Japan. Japan has the highestprevalence per million population, followed by the US.

The mortality rates associated with hemodialysis are striking andindicate that the life expectancy of patients entering into hemodialysisis markedly shortened. At every age, patients with ESRD on dialysis havesignificantly increased mortality when compared with nondialysispatients and individuals without kidney disease. At age 60 years, ahealthy person can expect to live for more than 20 years, whereas thelife expectancy of a 60-year-old patient starting hemodialysis is closerto 4 years (Aurora and Verelli, May 21, 2009. Chronic Renal Failure:Treatment & Medication. Emedicine.http://emedicine.medscape.com/article/238798-treatment).

International Patent Publication Nos. WO 2006/035434, WO 2008/104978, WO2008/106102, and WO 2009/001359 assigned to one of the assignees of thepresent invention relate to methods of treating acute kidney diseaseincluding acute renal failure following cardiac surgery.

Methods and compositions useful for treating CKD and for attenuatingprogression of CKD would be of great therapeutic value.

SUMMARY OF THE INVENTION

According to one aspect the present invention provides a method oftreating or preventing kidney damage in a subject at risk of chronickidney disease (CKD) associated with exposure to a recurrence of renalinsults comprising administering to the subject a therapeuticallyeffective dose of a compound which inhibits a target gene associatedwith kidney damage wherein the oligonucleotide compound is administeredto the subject within 24 hours of the renal insult. In some embodimentsthe compound is an oligonucleotide compound. In some embodiments theoligonucleotide compound inhibits expression of a target gene.

According to another aspect the present invention provides a method ofattenuating progression of chronic kidney disease (CKD) in a subject atrisk of CKD progression resulting from exposure to a recurrence of renalinsults comprising administering to the subject a therapeuticallyeffective dose of an oligonucleotide compound which down regulatesexpression of a target gene associated with kidney injury wherein theoligonucleotide compound is administered to the subject within 24 hoursof each renal insult. In some embodiments the kidney insult results inacute renal insult including acute kidney injury (AKI).

In various embodiments a subject at risk of chronic kidney disease (CKD)or CKD progression is a subject having any one or more of Type 1 or Type2 diabetes mellitus, high blood pressure (hypertension), highcholesterol, heart disease, liver disease, amyloidosis, Sickle celldisease, Systemic Lupus erythematosus, glomerulonephritis, polycystickidney disease, atherosclerosis, vascular diseases such as arteritis,vasculitis, or fibromuscular dysplasia or a subject that is about toundergo radiographical examination (i.e. administration of aradiocontrast agent) or a subject that uses nephrotoxic medications,including, without being limited to, analgesics such as acetaminophen(Tylenol®) and non-steroidal anti-inflammatory drugs (NSAIDs) (e.g.ibuprofen (Motrin®, Advil®)) that can cause analgesic nephropathy whenused regularly over long durations of time.

In another aspect the present invention relates to a method ofattentuating the severity of kidney damage resulting from renal insultin a subject suffering from chronic kidney disease (CKD) comprisingadministering to the subject a therapeutically effective dose of anoligonucleotide compound which inhibits expression of a target geneassociated with renal ischemia, thereby attenuating kidney damage. Insome embodiments kidney damage is AKI.

In other embodiments provided is a method to prevent progression of CKDresulting from acute kidney injury or insult in a subject suffering fromCKD comprising administering to the subject a therapeutically effectivedose of an oligonucleotide compound which inhibits expression of atarget gene associated with renal ischemia, thereby preventingprogression of CKD.

In some embodiments of the methods of the present invention, the renalinsult is selected from surgery including cardiovascular surgery,exposure to radiocontrast agent, myoglobinuria, ischemia/reperfusioninjury, urinary tract obstruction exposure to nephrotoxins, includingcontrast agents including radiocontrast agents; decreased perfusion;proteinuria; increased renal ammoniagenesis with interstitial injury;hyperlipidemia; hyperphosphatemia with calcium phosphate deposition. Insome embodiments the renal insult is selected from ischemia/reperfusion,sepsis and exposure to a radiocontrast agent.

In certain embodiments ischemia/reperfusion injury ensues during orfollowing cardiovascular surgery or cardiopulmonary surgery.Myoglobinurea results from myoglobin, which acts as an endogenousnephrotoxin by both direct proximal tubule cell (PTC) injury and renalvasoconstriction. A recurrence or plurality of renal insults refers to2, 3, 4, 5 or more renal insults of the same or different types.

In some embodiments the oligonucleotide is administered to the subjectwithin (i.e. prior to, simultaneously with or post) 72 hours, within 48hours, within 24 hours, within 16 hours, within 8 hours, within 4 hourspre or post renal insult and preferably at about 72 hours pre to aboutto about 8 hours post renal insult. In some embodiments theoligonucleotide is administered 0 to 4 hours post renal insult. In someembodiments the oligonucleotide is administered to the subject inproximity of the renal insult. in proximity refers to within one hour ofrenal insult, within 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2minutes or 1 minute post renal insult.

In various embodiments the oligonucleotide compound is selected from thegroup consisting of unmodified or chemically modified siRNA, shRNA, anaptamer, an antisense molecule, miRNA, and a ribozyme. In the presentlypreferred embodiments the inhibitor is chemically modified siRNA.

In some embodiments the target gene is a human gene whose expression isup regulated after renal insult. In some embodiments the target geneassociated with kidney injury, kidney damage, renal ischemia is selectedfrom a gene having an mRNA sequence set forth in Table 1, infra. Anon-limiting example of target genes include p53, tumor protein p53binding protein 2 (TP53BP2); leucine-rich repeats and death domaincontaining (LRDD); cytochrome b-245, alpha polypeptide (CYBA, p22phox);activating transcription factor 3 (ATF3); caspase 2, apoptosis-relatedcysteine peptidase (CASP2); NADPH oxidase 3 (NOX3); harakiri, BCL2interacting protein (HRK, BID3); complement component 1, q subcomponentbinding protein (C1QBP); BCL2/adenovirus E1B 19 kDa interacting protein3 (BNIP3); mitogen-activated protein kinase 8 (MAPK8, JNK1);mitogen-activated protein kinase 14 (MAPK14, p38); ras-related C3botulinum toxin substrate 1 (rho family, small GTP binding proteinRAC1); glycogen synthase kinase 3 beta (GSK3B); purinergic receptor P2X,ligand-gated ion channel, 7 (P2RX7); transient receptor potential cationchannel, subfamily M, member 2 (TRPM2); poly (ADP-ribose) glycohydrolase(PARG); CD38 molecule (CD38); STEAP family member 4 (STEAP4); bonemorphogenetic protein 2 (BMP2); gap junction protein, alpha 1, 43 kDa(connexin 43, GJA1); TYRO protein tyrosine kinase binding protein(TYROBP); connective tissue growth factor (CTGF); secretedphosphoprotein 1 (osteopontin, SPP1); ras homolog gene family, member A(RHOA); dual oxidase 1 (DUOX1), NOX1, NOX2 (gp91phox, CYBB), NOX4, NOX5,DUOX2 and associated proteins, NOXO1, NOXO2 (p47phox, NCF1) NOXA1, NOXA2(p67phox, NCF2) and p40phox (NCF4), ASPP1, CTDS, CAPNS1, REDD1, REDD2,HTRA2, KEAP1, SHC1, ZNHIT1, LGALS3, HI95, TGFb-1, ACE, MCP-1, CDK, MIF,ECE-1, ET-1, TSA, Smad2, Smad3, ALK5, STAT3, PTGDS, TLR2. In someembodiments the target gene is selected from p53 and CASP2. Withoutbeing bound to theory, any one or more of the aforementioned genes isupregulated by renal insult and inhibition of this upregulation of oneor more of those genes in the kidney protects the renal cells fromdamage including ischemic injury.

In another aspect the present invention provides a method of preventingthe development of chronic kidney disease (CKD) in a subject at risk ofdeveloping CKD resulting from exposure to a plurality of renal insultscomprising administering to the subject a therapeutically effective doseof an oligonucleotide compound which inhibits expression of a geneassociated with renal ischemia wherein the oligonucleotide compound isadministered to the subject within 16 hours of the renal insult.

In yet another aspect the present invention provides a method ofpreventing chronic kidney disease (CKD) from occurring in a subjectwhich may be predisposed or at risk of developing CKD resulting fromexposure to a plurality of renal insults comprising administering to thesubject a therapeutically effective dose of an oligonucleotide compoundwhich inhibits expression of a gene associated with renal ischemiawherein the oligonucleotide compound is administered to the subjectwithin 16 hours of the renal insult. Methods include sustained deliveryand controlled delivery for local or systemic delivery includingdelivery of siRNA using for example a delivery vehicle including pump, aslow or sustained release composition or an implant comprising a siRNAdepot. The delivery vehicle comprises natural and synthetic materials ora combination of natural and synthetic materials.

Kits for the treatment or prevention of chronic kidney disease (CKD) arealso provided. In some embodiments the invention provides a kit for thetreatment or prevention of chronic kidney disease (CKD) associated withexposure to a radiocontrast agent. In some embodiments a kit includes apackage containing a therapeutically effective dose of anoligonucleotide compound which inhibits expression of a gene associatedwith kidney damage in an amount effective to prevent radiocontrast agentinduced kidney damage and a radiocontrast agent in an amount effectiveto perform a radiographical examination. In certain embodiments anoligonucleotide compound is included as a separate individualpreparation and a radiocontrast agent as a separate individualpreparation. In some embodiments, an oligonucleotide compound and aradiocontrast agent are combined as a single composition. In someembodiments, an oligonucleotide compound preparation and a radiocontrastagent preparation are provided in different forms (e.g. one preparationis a liquid and the other preparation is a freeze-dried preparation). Insome embodiments, a kit further includes instructions for use.

In various embodiments the present invention provides a method employinga double stranded oligoribonucleotide compound that inhibits expressionof a target gene associated with AKI and progression to CKD. In variousembodiment the target gene is a gene associated withischemia/reperfusion injury (IRI). In certain preferred embodiments thetarget gene is selected from the genes listed in Table 1, set forthhereinbelow. In particular embodiments the double strandedoligoribonucleotide compounds are chemically modified siRNA.

In some embodiments the siRNA compound is chemically modified accordingto the following structure:

-   -   5′ (N)_(x)—Z 3′ (antisense strand)    -   3′Z′-(N′)_(y)-z″ 5′ (sense strand)        wherein each of N and N′ is a ribonucleotide which may be        unmodified or modified, or an unconventional moiety;        wherein each of (N)x and (N′)y is an oligonucleotide in which        each consecutive N or N′ is joined to the next N or N′ by a        covalent bond;        wherein Z and Z′ may be present or absent, but if present is        independently 1-5 consecutive nucleotides covalently attached at        the 3′ terminus of the strand in which it is present;        wherein z″ may be present or absent, but if present is a capping        moiety covalently attached at the 5′ terminus of (N′)y;        each of x and y is independently an integer between 18 and 40;        wherein the sequence of (N′)y is substantially complementary to        the sequence of (N)x; and wherein (N)x comprises an antisense        sequence substantially complementary to from about 18 to about        40 consecutive ribonucleotides present in an mRNA shown in Table        1, set forth in any one of SEQ ID NOS:1-115.

In some embodiments the covalent bond joining each consecutive N or N′is a phosphodiester bond. In various embodiments all the covalent bondsare phosphodiester bonds.

In various embodiments the compound comprises ribonucleotides whereinx=y and each of x and y is 19, 20, 21, 22 or 23. In some embodimentsx=y=23. In other embodiments x=y=19.

In some embodiments the compound is blunt ended, for example whereinboth Z and Z′ are absent. In an alternative embodiment, the compoundcomprises at least one 3′ overhang, wherein at least one of Z or Z′ ispresent. Z and Z′ can independently comprise one or more covalentlylinked modified or non-modified nucleotides, for example inverted dT ordA; dT, LNA, mirror nucleotide and the like. In some embodiments each ofZ and Z′ are independently selected from dT and dTdT.

In some embodiments each of (N)x and (N′)y consist of unmodifiednucleotides.

In some embodiments N or N′ comprises a modification in the sugarresidue of one or more ribonucleotides. In other embodiments thecompound comprises at least one ribonucleotide modified in the sugarresidue. In some embodiments the compound comprises a modification atthe 2′ position of the sugar residue. In some embodiments themodification in the 2′ position comprises the presence of an amino, afluoro, an alkoxy or an alkyl moiety. In certain embodiments the 2′modification comprises a methoxy moiety (also known as 2′-O-methyl;2′-O-Me; 2′-O—CH₃). In some embodiments in each of (N)x and (N′)y theribonucleotides alternate between modified ribonucleotides andunmodified ribonucleotides each modified ribonucleotide being modifiedso as to have a 2′-O-methyl on its sugar and the ribonucleotide locatedat the middle position of (N)x being unmodified and the ribonucleotidelocated at the middle position of (N′)y being modified. In someembodiments the preferred compound is I5, which targets p53.

In some embodiments the siRNA compound comprises modified alternatingribonucleotides in one or both of the antisense and the sense strands.In other embodiments the compound comprises modified alternatingribonucleotides in the antisense strand (N)x only. In certainembodiments the middle ribonucleotide of the antisense strand is notmodified; e.g. ribonucleotide in position 10 in a 19-mer strand orposition 12 in a 23-mer strand.

In additional embodiments the compound comprises modifiedribonucleotides in alternating positions wherein each N at the 5′ and 3′termini of (N)_(x) are modified in their sugar residues, and each N′ atthe 5′ and 3′ termini of (N′)_(y) are unmodified in their sugarresidues. In some embodiments, neither (N)_(x) nor (N′)_(y) arephosphorylated at the 3′ and 5′ termini. In other embodiments either orboth (N)_(x) and (N′)_(y) are phosphorylated at the 3′ termini.

In some embodiments (N)x comprises modified and unmodifiedribonucleotides, each modified ribonucleotide having a 2′-O-methyl onits sugar, wherein N at the 3′ terminus of (N)x is a modifiedribonucleotide, (N)x comprises at least five alternating modifiedribonucleotides beginning at the 3′ end and at least nine modifiedribonucleotides in total and each remaining N is an unmodifiedribonucleotide. In some embodiments, (N)x comprises 2′O Me modifiedribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17 and 19. In otherembodiments (N)x comprises 2′O Me modified ribonucleotides at positions1, 3, 5, 7, 9, 11, 13, 15, 17 and 19.

In some embodiments the unconventional moiety is selected from a mirrornucleotide and a nucleotide joined to an adjacent nucleotide by a 2′-5′internucleotide phosphate bond. In some embodiments the mirrornucleotide is selected from an L-ribonucleotide (L-RNA) and anL-deoxyribonucleotide (L-DNA). In some embodiments (N′)y comprises atleast one unconventional moiety.

In one embodiment of the above structure, the compound comprises atleast one mirror nucleotide at one or both termini in (N′)y. In variousembodiments the compound comprises two consecutive mirror nucleotides,one at the 3′ penultimate position and one at the 3′ terminus in (N′)y.In one preferred embodiment x=y=19 and (N′)y comprises an L-DNA atposition 18.

In some embodiments x=y=19 and (N′)y, consists of unmodifiedribonucleotides at positions 1-17 and 19 and one L-DNA at the 3′penultimate position (position 18). In other embodiments x=y=19 and(N′)y consists of unmodified ribonucleotides at position 1-16 and 19 andtwo consecutive L-DNA at the 3′ penultimate position (positions 17 and18).

In another embodiment of the above structure, (N′)y further comprisesone or more nucleotides containing an intra-sugar bridge at one or bothtermini.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a proposed vicious circle of mutual reinforcementbetween AKI and CKD in the development of ESRD (from: B. Molitoris(2008) “Contrast nephropathy: are short-term outcome measures adequatefor quantification of long-term renal risk”, Nat Clin Pract Nephrol.2008 4:594-5.)

FIG. 2 provides an outline of the study design as described in Example2, hereinbelow.

FIG. 3 shows the effect of p53 siRNA on kidney function followingrepetitive ischemic injury.

FIG. 4 shows that siP53 protects GFR and minimizes proteinuria.

FIG. 5. Effect of siP53 (12 mg/kg) on histology after five monthlycycles of ischemic injury.

FIG. 6 provides the study design for a CKD model established bynephrectomy and multiple AKI induction at bimonthly intervals. CKD wasinduced by subjecting rats to uninephrectomy and multiple AKI (3-4 overa period of 7-8 months) and feeding them with high salt (Na, sodium)diet.

FIG. 7 shows pretreatment kidney function parameters after 7-8 months offeeding either a high or low Na diet. Serum creatinine, GFR and Urineprotein were similar in rats treated with QM5 siRNA or carrier only.High Na diet resulted in more rapid progression of CKD, loss of GFR butnot proteinuria.

FIG. 8 shows the efficacy of QM5 (siP53) on prevention of AKI in CKDinduced animals.

FIGS. 9A-9H show the histopatholgy evaluation at termination of the CKDstudy outlined in FIG. 6 and described in Example 2.2. FIGS. 9A-9Crelate to acute injury parameters tubular necrosis (9A), tubulardialation (9B) and casts (9C). FIGS. 9D-9H relate to chronic injuryparameters glomerular damage (9D), interstitial cellular infiltrate(9E); interstitial fibrosis (9F), tubular atrophy or dilation (9G) andvasculopathy (9H).

FIG. 10 shows results of histopathogy evaluation at termination andrecords the average acute and chronic injury scores in treated vsuntreated group.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates in general to a method of attenuating theprogression of chronic kidney disease or preventing exacerbation of CKDprogression in a subject at risk thereof. The method employs generallycompounds which down-regulate expression of various target genesassociated with acute kidney injury and in particular with ischemicreperfusion injury. The method employs chemically modified smallinterfering RNA oligonucleotides (siRNAs), possessing structures andmodifications which may increase activity, increase stability, and orminimize toxicity, reduce off target effect or reduce innate immuneresponse when compared to the unmodified compound.

Table 1, below, sets forth the gene identification number (gi) with anNCBI accession number for non-limiting examples of target genes. Thetable sets forth the respective mRNA sequences, the gi number (geneidentifier number) and the sequence identifier number (SEQ ID NO) forthe corresponding mRNA.

TABLE 1 Non-limiting list of target genes Gene Full name and Human GeneID REDD1 DDIT4, DNA-damage-inducible transcript 4gi|56676369|ref|NM_019058 (SEQ ID NO: 1) REDD2 DNA-damage-inducibletranscript 4-like gi|34222182|ref|NM_145244 (SEQ ID NO: 2) TP53BP2 tumorprotein p53 binding protein, 2 (ASPP2) gi|112799848|ref|NM_001031685.2(SEQ ID NO: 3) gi|112799845|ref|NM_005426.2 (SEQ ID NO: 4): LRDDleucine-rich repeats and death domain containinggi|61742781|ref|NM_018494.3 (SEQ ID NO: 5) gi|61742783|ref|NM_145886.2(SEQ ID NO: 6) gi|61742785|ref|NM_145887.2 (SEQ ID NO: 7) CYBAcytochrome b-245, alpha polypeptide gi|68509913|ref|NM_000101.2|(SEQ IDNO: 8) ATF3 activating transcription factor 3gi|95102484|ref|NM_001030287.2| v. 3 (SEQ ID NO: 9)gi|71902534|ref|NM_001674.2|v. 1 (SEQ ID NO: 10)gi|95102482|ref|NM_001040619.1| (SEQ ID NO: 11) CASP2 caspase 2,apoptosis-related cysteine peptidase gi|39995058|ref|NM_032982.2 (SEQ IDNO: 12) gi|39995060|ref|NM_032983.2 (SEQ ID NO: 13) NOX3 NADPH oxidase 3Gi|11136625|ref|NM_015718.1 (SEQ ID NO: 14) HRK harakirigi|4504492|ref|NM_003806.1 (SEQ ID NO: 15) C1QBP complement component 1,q subcomponent binding protein gi|28872801|ref|NM_001212.3 (SEQ ID NO:16) BNIP3 BCL2/adenovirus E1B 19 kDa interacting protein 3Gi|7669480|ref|NM_004052.2 (SEQ ID NO: 17) MAPK8 mitogen-activatedprotein kinase 8 gi|20986493|ref|NM_002750.2 v. 2 (SEQ ID NO: 18)gi|20986522|ref|NM_139049.1 v. 1 (SEQ ID NO: 19)gi|20986518|ref|NM_139046.1 v. 3 (SEQ ID NO: 20)gi|20986520|ref|NM_139047.1 v. 4 (SEQ ID NO: 21) MAPK14mitogen-activated protein kinase 14 gi|20986511|ref|NM_139012.1 v. 2(SEQ ID NO: 22) gi|20986515|ref|NM_139014.1 v. 4 (SEQ ID NO: 23)gi|4503068|ref|NM_001315.1 v. 1 (SEQ ID NO: 24)gi|20986513|ref|NM_139013.1 v. 3 (SEQ ID NO: 25) Rac1 ras-related C3botulinum toxin substrate 1 (rho family, small GTP binding protein)gi|156071511|ref|NM_018890.3(SEQ ID NO: 26)gi|156071503|ref|NM_006908.4(SEQ ID NO: 27) GSK3B glycogen synthasekinase 3 beta gi|21361339|ref|NM_002093.2(SEQ ID NO: 28)gi|225903436|ref|NM_001146156.1 (SEQ ID NO: 29) P2RX7 purinergicreceptor P2X, ligand-gated ion channel, 7 gi|34335273|ref|NM_002562.4(SEQ ID NO: 30) TRPM2 transient receptor potential cation channel,subfamily M, member 2 gi|67906812|ref|NM_003307.3 v. L (SEQ ID NO: 31)PARG poly (ADP-ribose) glycohydrolase gi|70610135|ref|NM_003631.2 (SEQID NO: 32) CD38 CD38 molecule Gi|38454325|ref|NM_001775.2 (SEQ ID NO:33) STEAP4 STEAP family member 4 Gi|13375867|ref|NM_024636.1 (SEQ ID NO:34) BMP2 bone morphogenetic protein 2 gi|80861484|ref|NM_001200.2(SEQ IDNO: 35) GJA1 gap junction protein, alpha 1, 43 kDagi|4755136|ref|NM_000165.2(SEQ ID NO: 36) TYROBP TYRO protein tyrosinekinase binding protein gi|291045273|ref|NM_001173515.1 variant 4 (SEQ IDNO: 37) gi|291045270|ref|NM_198125.2| variant 2 (SEQ ID NO: 38)gi|291045269|ref|NM_003332.3| variant 1 (SEQ ID NO: 39)gi|291045271|ref|NM_001173514.1 variant 3 (SEQ ID NO: 40) CTGFconnective tissue growth factor gi|4503122|ref|NM_001901.1(SEQ ID NO:41) SPP1 secreted phosphoprotein 1 gi|91206461|ref|NM_001040058.1(SEQ IDNO: 42) gi|38146097|ref|NM_000582.2 (SEQ ID NO: 43)gi|91598938|ref|NM_001040060.1(SEQ ID NO: 44) RHOA ras homolog genefamily member A gi|50593005|ref|NM_001664.2(SEQ ID NO: 45) DUOX1 dualoxidase 1 gi|28872749|ref|NM_017434.3 (SEQ ID NO: 46)gi|28872750|ref|NM_175940.1 (SEQ ID NO: 47) NOX4 NADPH oxidase 4gi|219842344|ref|NM_016931.3| v. 1 (SEQ ID NO: 48)gi|219842345|ref|NM_001143836.1| v. 1 (SEQ ID NO: 49)gi|219842347|ref|NM_001143837.1| v. 1 (SEQ ID NO: 50) NOX1 NADPH oxidase1 (gi:21614529, NM_007052 isoform 1L; SEQ ID NO: 51) (gi:7669509,NM_013955 isoform 1Lv; SEQ ID NO: 52) NOX2 NADPH oxidase 2 (gp91pho,(gi:6996020, NM_000397; SEQ ID NO: 53) CYBB) NOX5 NADPH oxidase 5(gi:20127623, NM_024505; SEQ ID NO: 54) DUOX2 Dual oxidase 2(gi:132566531, NM_014080; SEQ ID NO: 55) NOXO1 NADPH oxidase organizer 1(gi:34222190, variant a, NM_144603, SEQ ID NO: 56) (gi:41281810, variantb, NM_172167; SEQ ID NO: 57) (gi:41281827, variant c, NM_172168; SEQ IDNO: 58) NCF1 NADPH oxidase organizer 2 (p47phox, (gi:115298671,NM_000265; SEQ ID NO: 59) NOXO2) NOXA1 NADPH oxidase activator 1(gi:41393186, NM_006647; SEQ ID NO: 60) NCF2 NADPH oxidase activator 2(p67phox, (gi:67189969, NM_000433; SEQ ID NO: 61) NOXA2)(gi|189083741|ref|NM_001127651.1| v. 2 (SEQ ID NO: 62) ASPP1 proteinphosphatase 1, regulatory (inhibitor) subunit 13B (PPP1R13B)gi|121114286|ref|NM_015316.2| (SEQ ID NO: 63) CTSD Cathepsin Dgi|23110949|ref|NM_001909.3| (SEQ ID NO: 64) CAPNS1 Calpain smallsubunit 1 gi|51599152|ref|NM_001749.2| Variant 1 (SEQ ID NO: 65)gi|51599150|ref|NM_001003962.1| variant 2 (SEQ ID NO: 66) p53 (TP53)tumor protein p53 gi|187830767|ref|NM_000546.4| variant 1 (SEQ ID NO:67) gi|187830776|ref|NM_001126112.1| variant 2(SEQ ID NO: 68)gi|187830854|ref|NM_001126114.1| variant 3(SEQ ID NO: 69)gi|187830822|ref|NM_001126113.1| variant 4(SEQ ID NO: 70)gi|187830893|ref|NM_001126115.1| variant 5(SEQ ID NO: 71)gi|187830900|ref|NM_001126116.1| variant 6(SEQ ID NO: 72)gi|187830908|ref|NM_001126117.1| variant 7(SEQ ID NO: 73) HTRA2 Htraserine peptidase 2 var 1 gi:73747817, NM_013247 (SEQ ID NO: 74) var 2gi:73747818, NM_145074 (SEQ ID NO: 75) KEAP1 Kelch-like ECH-associatedprotein 1 var 1 gi:45269144 NM_203500 (SEQ ID NO: 76) var 2 gi:45269143NM_012289 (SEQ ID NO: 77) SHC1 Src homology 2 domain containing)transforming prot. 1 gi|194239661|ref|NM_183001.4| (SEQ ID NO: 78gi|194239660|ref|NM_003029.4| (SEQ ID NO: 79)gi|194239663|ref|NM_001130040.1| (SEQ ID NO: 80)gi|194239667|ref|NM_001130041.1| (SEQ ID NO: 81) ZNHIT1 Zn finger HITtype 1 gi:37594439|; NM_006349 (SEQ ID NO: 82) LGALS3 lectingalactoside-binding soluble 3 var 1 gi:115430222 NM_002306 (SEQ ID NO:83) var 2 gi:115430224 NR_003225 (SEQ ID NO: 84) var 3gi|294345474|ref|NM_001177388.1| (SEQ ID NO: 85) HI95 Sestrin2gi:32454742 NM_031459 (SEQ ID NO: 86) TGFb-1 transforming growth factorbeta-1 gi: 63025221 NM_000660 (SEQ ID NO: 87) ACE angiotensin-convertingenzyme transcript variant 2: gi|23238213|ref|NM_152830.1| (SEQ ID NO:88) transcript variant 1, gi|23238217|ref|NM_000789.2| (SEQ ID NO: 89)CCL2 Homo sapiens chemokine (C-C motif) ligand 2 (CCL2), mRNAgi|56119169|ref|NM_002982.3| (SEQ ID NO: 90) CDK1 Homo sapiens celldivision cycle 2, G1 to S and G2 to M (CDC2), (CDC2)gi|195927038|ref|NM_001786.3| v. 1 (SEQ ID NO: 91)gi|195927039|ref|NM_033379.3| v. 2 (SEQ ID NO: 92)gi|195927040|ref|NM_001130829.1| v. 3 (SEQ ID NO: 93) MIF macrophageinhibitory factor gi:4505184 NM_002415 (SEQ ID NO: 94) ECE-1 endothelinconverting enzyme gi|164519130|ref|NM_001397.2| variant 1, (SEQ ID NO:95) gi|164519139|ref|NM_001113349.1| (SEQ ID NO: 96)gi|164519135|ref|NM_001113347.1| (SEQ ID NO: 97)gi|164519137|ref|NM_001113348.1| (SEQ ID NO: 98) ET-1 (EDN1) Homosapiens endothelin 1, mRNA gi|154800436|ref|NM_001955.3| (SEQ ID NO: 99)TSA (LY6E) (Thymic shared antigen1) Homo sapiens lymphocyte antigen 6complex, locus E (LY6E), mRNA variant 1, gi|187827163|ref|NM_002346.2|(SEQ ID NO: 100) variant 2, gi|187761330|ref|NM_001127213.1| (SEQ ID NO:101) Smad2 Homo sapiens SMAD family member 2 (SMAD2), mRNA variant 1,gi|118572580|ref|NM_005901.4| (SEQ ID NO: 102) variant 2,gi|118572581|ref|NM_001003652.2 (SEQ ID NO: 103)gi|209693425|ref|NM_001135937.1| var 3 (SEQ ID NO: 104) Smad3 Homosapiens SMAD family member 3 (SMAD3), mRNA variant 1,gi|52352808|ref|NM_005902.3 (SEQ ID NO: 105)gi|223029439|ref|NM_001145102.1| v. 2 (SEQ ID NO: 106)gi|223029441|ref|NM_001145103.1| v. 3 (SEQ ID NO: 107)gi|223029443|ref|NM_001145104.1| v. 4 (SEQ ID NO: 108) TGFBR1 Homosapiens transforming growth factor, beta receptor 1, mRNA (ALK5,activin-receptor like kinase) transcript variant 1,gi|66346739|ref|NM_004612.2| (SEQ ID NO: 109) ranscript variant 2,gi|195963411|ref|NM_001130916.1| (SEQ ID NO: 110) STAT3 Homo sapienssignal transducer and activator of transcription 3 (acute- phaseresponse factor) (STAT3), mRNA transcript variant 1,gi|47080104|ref|NM_139276.2| (SEQ ID NO: 111) transcript variant 2gi|47080105|ref|NM_003150.3| (SEQ ID NO: 112) transcript variant 3gi|47458819|ref|NM_213662.1| (SEQ ID NO: 113) PTGDS Homo sapiensprostaglandin D2 synthase 21 kDa (brain) (PTGDS), mRNAgi|38505192|ref|NM_000954.5| (SEQ ID NO: 114) TLR2 Homo sapienstoll-like receptor 2 (TLR2), mRNA gi|68160956|ref|NM_003264.3| (SEQ IDNO: 115)

DEFINITIONS

For convenience certain terms employed in the specification, examplesand claims are described herein.

It is to be noted that, as used herein, the singular forms “a”, “an” and“the” include plural forms unless the content clearly dictatesotherwise.

Where aspects or embodiments of the invention are described in terms ofMarkush groups or other grouping of alternatives, those skilled in theart will recognize that the invention is also thereby described in termsof any individual member or subgroup of members of the group.

An “inhibitor” is a compound, which is capable of reducing (partially orfully) or down regulating the expression of a gene or the activity ofthe product of such gene to an extent sufficient to achieve a desiredbiological or physiological effect. The term “inhibitor” as used hereinrefers to one or more of an oligonucleotide inhibitor, including siRNA,shRNA, synthetic shRNA; miRNA, antisense RNA and DNA and ribozymes.

A “siRNA inhibitor” is a compound which is capable of reducing theexpression of a gene or the activity of the product of such gene to anextent sufficient to achieve a desired biological or physiologicaleffect. The term “siRNA inhibitor” as used herein refers to one or moreof a siRNA, shRNA, synthetic shRNA; miRNA. Inhibition may also bereferred to as down-regulation or, for RNAi, silencing.

The term “inhibit” or “down regulate” as used herein refers to reducingthe expression of a gene or the activity of the product of such gene toan extent sufficient to achieve a desired biological or physiologicaleffect. Inhibition or down regulation may be complete or partial.

As used herein, the term “inhibition” or “down-regulation” of a targetgene means reduction of the gene expression (transcription ortranslation) or polypeptide activity of a gene selected from the groupconsisting of any one of SEQ ID NO: 1-115 or an SNP (single nucleotidepolymorphism) or other variants thereof. The gi number for the mRNA ofeach target gene is set forth in Table 1. The polynucleotide sequence ofthe target mRNA sequence, refers to the mRNA sequences set forth herein,or any homologous sequences thereof preferably having at least 70%identity, more preferably 80% identity, even more preferably 90% or 95%identity to any one of mRNA set forth herein. Therefore, polynucleotidewhich have undergone mutations, alterations or modifications asdescribed herein are encompassed in the present invention. The terms“mRNA polynucleotide sequence” and “mRNA” are used interchangeably.

As used herein, the terms “polynucleotide” and “nucleic acid” may beused interchangeably and refer to nucleotide sequences comprisingdeoxyribonucleic acid (DNA), and ribonucleic acid (RNA). The termsshould also be understood to include, as equivalents, analogs of eitherRNA or DNA made from nucleotide analogs. Throughout this application,mRNA sequences are set forth as representing the corresponding genes.

“Oligonucleotide” or “oligomer” refers to a deoxyribonucleotide orribonucleotide sequence from about 2 to about 50 nucleotides. Each DNAor RNA nucleotide may be independently natural or synthetic, and ormodified or unmodified. Modifications include changes to the sugarmoiety, the base moiety and or the linkages between nucleotides in theoligonucleotide. The compounds of the present invention encompassmolecules comprising deoxyribonucleotides, ribonucleotides, modifieddeoxyribonucleotides, modified ribonucleotides and combinations thereof.

“Nucleotide” is meant to encompass deoxyribonucleotides andribonucleotides, which may be natural or synthetic, and or modified orunmodified. Modifications include changes to the sugar moiety, the basemoiety and or the linkages between ribonucleotides in theoligoribonucleotide. As used herein, the term “ribonucleotide”encompasses natural and synthetic, unmodified and modifiedribonucleotides. Modifications include changes to the sugar moiety, tothe base moiety and/or to the linkages between ribonucleotides in theoligonucleotide.

Analogs of, or modifications to, a nucleotide/oligonucleotide arepreferably employed with the present invention, provided that saidanalog or modification does not substantially adversely affect thefunction of the nucleotide/oligonucleotide. In some embodiments achemical modification results in an increase in activity or stability ora reduction in off-target effects or induction of innate immuneresponses. Acceptable modifications include modifications of the sugarmoiety, modifications of the base moiety, modifications in theinternucleotide linkages and combinations thereof.

The nucleotides can be selected from naturally occurring or syntheticmodified bases. Naturally occurring bases include adenine, guanine,cytosine, thymine and uracil. Modified bases of nucleotides includeinosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl andother alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine,8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyladenine and other 8-substituted adenines, 8-halo guanines, 8-aminoguanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine andother substituted guanines, other aza and deaza adenines, other aza anddeaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.

In addition, compounds comprising nucleotide analogs can be preparedwherein the structure of one or more nucleotide is fundamentally alteredand better suited as therapeutic or experimental reagents. An example ofa nucleotide analog is a peptide nucleic acid (PNA) wherein thedeoxyribose (or ribose) phosphate backbone in DNA (or RNA is replacedwith a polyamide backbone which is similar to that found in peptides.PNA analogs have been shown to be resistant to enzymatic degradation andto have extended lives in vivo and in vitro.

Possible modifications to the sugar residue are manifold and include2′-O alkyl, locked nucleic acid (LNA), glycol nucleic acid (GNA),threose nucleic acid (TNA), arabinoside, altritol (ANA) and other,6-membered sugars including morpholinos, and cyclohexinyls.

LNA compounds are disclosed in International Patent Publication Nos. WO00/47599, WO 99/14226, and WO 98/39352. Examples of siRNA compoundscomprising LNA nucleotides are disclosed in Elmen et al., (NAR 2005.33(1):439-447) and in International Patent Publication No. WO2004/083430.

Backbone modifications, such as ethyl (resulting in a phospho-ethyltriester); propyl (resulting in a phospho-propyl triester); and butyl(resulting in a phospho-butyl triester) are also possible. Otherbackbone modifications include polymer backbones, cyclic backbones,acyclic backbones, thiophosphate-D-ribose backbones, amidates,phosphonoacetate derivatives. Certain structures include siRNA compoundshaving one or a plurality of 2′-5′ internucleotide linkages (bridges orbackbone).

Additional modifications which may be present in the molecules of thepresent invention include nucleoside modifications such as artificialnucleic acids, peptide nucleic acid (PNA), morpholino and locked nucleicacid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA),arabinoside, and mirror nucleoside (for example, beta-L-deoxynucleosideinstead of beta-D-deoxynucleoside). Further, said molecules mayadditionally contain modifications on the sugar, such as 2′-alkyl,2′-fluoro, 2′-deoxy-2′-fluoro, 2′ O-allyl, 2′-amine and 2′-alkoxy.Additional sugar modifications are discussed herein.

Further, the inhibitory nucleic acid molecules of the present inventionmay comprise one or more gaps and/or one or more nicks and/or one oremore mismatches. Without wishing to be bound by theory, gaps, nicks andmismatches have the advantage of partially destabilizing the nucleicacid/siRNA, so that it may be more easily processed by endogenouscellular machinery such as DICER, DROSHA or RISC into its inhibitorycomponents.

In the context of the present invention, a gap in a nucleic acid refersto the absence of one or more internal nucleotides in one strand, whilea nick in a nucleic acid refers to the absence of an internucleotidelinkage between two adjacent nucleotides in one strand. Any of themolecules of the present invention may contain one or more gaps and/orone or more nicks.

siRNAs and RNA Interference

RNA interference (RNAi) is a phenomenon involving double-stranded (ds)RNA-dependent gene specific posttranscriptional silencing. Originally,attempts to study this phenomenon and to manipulate mammalian cellsexperimentally were frustrated by an active, non-specific antiviraldefense mechanism which was activated in response to long dsRNAmolecules (Gil et al. Apoptosis, 2000. 5:107-114). Later it wasdiscovered that synthetic duplexes of 21 nucleotide RNAs could mediategene specific RNAi in mammalian cells, without the stimulation of thegeneric antiviral defense mechanisms (see Elbashir et al. Nature 2001,411:494-498 and Caplen et al. PNAS USA 2001, 98:9742-9747). As a result,small interfering RNAs (siRNAs), which are short double-stranded RNAs,have become powerful tools in attempting to understand gene function.Thus RNA interference (RNAi) refers to the process of sequence-specificpost-transcriptional gene silencing in mammals mediated by smallinterfering RNAs (siRNAs) (Fire et al, Nature 1998. 391, 806) ormicroRNAs (miRNA; Ambros, Nature 2004 431:7006, 350-55; and Bartel,Cell. 2004. 116(2):281-97). The corresponding process in plants iscommonly referred to as specific post transcriptional gene silencing orRNA silencing and is referred to as quelling in fungi.

A siRNA is a double-stranded RNA molecule which inhibits or downregulates, either partially or fully, a gene or the expression of agene/mRNA of its endogenous or cellular counterpart, or of an exogenousgene such as a viral nucleic acid. The mechanism of RNA interference isdetailed infra.

Several studies have revealed that siRNA therapeutics are effective invivo in both mammals and in humans. Bitko et al., have shown thatspecific siRNA molecules directed against the respiratory syncytialvirus (RSV) nucleocapsid N gene are effective in treating mice whenadministered intranasally (Bitko et al., Nat. Med. 2005, 11(1):50-55).siRNA has recently been successfully used for inhibition in primates(Tolentino et al., Retina 2004. 24(1):132-138). For a review of the useof siRNA as therapeutics, see for example Barik (J. Mol. Med. 2005. 83:764-773) or Dykxhoorn et al (2006. Gene Ther. 13:541-552).

siRNA Structures

The selection and synthesis of siRNA corresponding to known genes hasbeen widely reported; (see for example Ui-Tei et al., J Biomed Biotech.2006; 2006: 65052; Chalk et al., BBRC. 2004, 319(1): 264-74; Sioud &Leirdal, Met. Mol Biol.; 2004, 252:457-69; Levenkova et al., Bioinform.2004, 20(3):430-2; Ui-Tei et al., NAR. 2004, 32(3):936-48).

For examples of the use of, and production of, modified siRNA see, forexample, Braasch et al., Biochem. 2003, 42(26):7967-75; Chiu et al.,RNA, 2003, 9(9):1034-48; PCT publications WO 2004/015107 (atugen AG) andWO 02/44321 (Tuschl et al). U.S. Pat. Nos. 5,898,031 and 6,107,094,teach chemically modified oligomers. US Patent Publication Nos.2005/0080246 and 2005/0042647 relate to oligomeric compounds having analternating motif and dsRNA compounds having chemically modifiedinternucleoside linkages, respectively.

Other modifications have been disclosed. The inclusion of a 5′-phosphatemoiety was shown to enhance activity of siRNAs in Drosophila embryos(Boutla, et al., Curr. Biol. 2001, 11:1776-1780) and is required forsiRNA function in human HeLa cells (Schwarz et al., Mol. Cell, 2002,10:537-48). Amarzguioui et al., (NAR, 2003, 31(2):589-95) showed thatsiRNA activity depended on the positioning of the 2′-O-methylmodifications. Holen et al (NAR. 2003, 31(9):2401-07) report that ansiRNA having small numbers of 2′-O-methyl modified nucleosides gave goodactivity compared to wild type but that the activity decreased as thenumbers of 2′-O-methyl modified nucleosides was increased. Chiu and Rana(RNA. 2003, 9:1034-48) teach that incorporation of 2′-O-methyl modifiednucleosides in the sense or antisense strand (fully modified strands)severely reduced siRNA activity relative to unmodified siRNA. Theplacement of a 2′-O-methyl group at the 5′-terminus on the antisensestrand was reported to severely limit activity whereas placement at the3′-terminus of the antisense and at both termini of the sense strand wastolerated (Czauderna et al., NAR. 2003, 31(11):2705-16; WO 2004/015107).The molecules of the present invention offer an advantage in that theyare non-toxic and may be formulated as pharmaceutical compositions fortreatment of various diseases.

International Patent Publication No. WO 2008/050329 to the assignee ofthe present invention and hereby incorporated in its entirely relates tosiRNA compounds, compositions comprising same and to methods of usethereof for treating diseases and disorders related to expression ofproapoptotic genes. U.S. Ser. No. 11/655,610 relates to methods oftreating hearing impairment by inhibiting a pro-apoptotic gene ingeneral and p53 in particular.

Oligonucleotides

The present invention provides methods employing oligonucleotideinhibitors including double-stranded oligonucleotides (e.g. siRNA),which down-regulate the expression of a desired gene. A siRNA is aduplex oligoribonucleotide in which the sense strand is derived from themRNA sequence of the desired gene, and the antisense strand iscomplementary to the sense strand. In general, some deviation from thetarget mRNA sequence is tolerated without compromising the siRNAactivity (see e.g. Czauderna et al., NAR. 2003, 31(11):2705-2716).Without being bound by theory, an siRNA of the invention inhibits geneexpression on a post-transcriptional level with or without destroyingthe mRNA; siRNA may target the mRNA for specific cleavage anddegradation and/or may inhibit translation from the targeted message.

In various embodiments the siRNA comprises an RNA duplex comprising afirst strand and a second strand, whereby the first strand comprises aribonucleotide sequence at least partially complementary to about 18 toabout 40 consecutive nucleotides of a target nucleic acid which is mRNAtranscribed from a target gene, and the second strand comprises aribonucleotide sequence at least partially complementary to the firststrand and wherein said first strand and or said second strand comprisesa one or more chemically modified ribonucleotides and or unconventionalmoieties.

In one embodiment the siRNA compound comprises at least oneribonucleotide comprising a 2′ modification on the sugar moiety(“2′sugar modification”). In certain embodiments the compound comprises2′O-alkyl or 2′-fluoro or 2′O-allyl or any other 2′ modification,optionally on alternate positions. Other stabilizing modifications arealso possible (e.g. terminal modifications). In some embodiments apreferred 2′O-alkyl is 2′O-methyl (methoxy, 2′OMe) sugar modification.

In some embodiments the backbone of the oligonucleotides is modified andcomprises phosphate-D-ribose entities but may also containthiophosphate-D-ribose entities, triester, thioate, 2′-5′ bridgedbackbone (also may be referred to as 5′-2′), PACE and the like.

As used herein, the terms “non-pairing nucleotide analog” means anucleotide analog which comprises a non-base pairing moiety includingbut not limited to: 6 des amino adenosine (Nebularine), 4-Me-indole,3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me riboU, N3-Me riboT,N3-Me-dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me-dC. In someembodiments the non-base pairing nucleotide analog is a ribonucleotide.In other embodiments it is a deoxyribonucleotide. In addition, analoguesof polynucleotides may be prepared wherein the structure of one or morenucleotide is fundamentally altered and better suited as therapeutic orexperimental reagents. An example of a nucleotide analogue is a peptidenucleic acid (PNA) wherein the deoxyribose (or ribose) phosphatebackbone in DNA (or RNA) is replaced with a polyamide backbone which issimilar to that found in peptides. PNA analogues have been shown to beresistant to enzymatic degradation and to enhance stability in vivo andin vitro. Other useful modifications include polymer backbones, cyclicbackbones, acyclic backbones, thiophosphate-D-ribose backbones, triesterbackbones, thioate backbones, 2′-5′ bridged backbone, artificial nucleicacids, morpholino nucleic acids, glycol nucleic acid (GNA), threosenucleic acid (TNA), arabinoside, and mirror nucleoside (for example,beta-L-deoxyribonucleoside instead of beta-D-deoxyribonucleoside). Thecompounds of the present invention can be synthesized using one or moreinverted nucleotides, for example inverted thymidine or inverted adenine(see, for example, Takei, et al., 2002, JBC 277(26):23800-06).

Additional modifications include terminal modifications on the 5′ and/or3′ part of the oligonucleotides and are also known as capping moieties.Such terminal modifications are selected from a nucleotide, a modifiednucleotide, a lipid, a peptide, a sugar and an inverted abasic moiety.

What is sometimes referred to in the present invention as an “abasicnucleotide” or “abasic nucleotide analog” is more properly referred toas a pseudo-nucleotide or an unconventional moiety. A nucleotide is amonomeric unit of nucleic acid, consisting of a ribose or deoxyribosesugar, a phosphate, and a base (adenine, guanine, thymine, or cytosinein DNA; adenine, guanine, uracil, or cytosine in RNA). A modifiednucleotide comprises a modification in one or more of the sugar,phosphate and or base. The abasic pseudo-nucleotide lacks a base, andthus is not strictly a nucleotide.

The term “unconventional moiety” as used herein refers to abasic ribosemoiety, an abasic deoxyribose moiety, a deoxyribonucleotide, a modifieddeoxyribonucleotide, a mirror nucleotide, a non-base pairing nucleotideanalog and a nucleotide joined to an adjacent nucleotide by a 2′-5′internucleotide phosphate bond; bridged nucleic acids including LNA andethylene bridged nucleic acids. In some embodiments of the presentinvention a preferred unconventional moiety is an abasic ribose moiety,an abasic deoxyribose moiety, a deoxyribonucleotide, a mirrornucleotide, and a nucleotide joined to an adjacent nucleotide by a 2′-5′internucleotide phosphate bond.

Abasic deoxyribose moiety includes for example abasicdeoxyribose-3′-phosphate; 1,2-dideoxy-D-ribofuranose-3-phosphate;1,4-anhydro-2-deoxy-D-ribitol-3-phosphate. Inverted abasic deoxyribosemoiety includes inverted deoxyriboabasic; 3′,5′ inverted deoxyabasic5′-phosphate.

A “mirror” nucleotide is a nucleotide with reversed chirality to thenaturally occurring or commonly employed nucleotide, i.e., a mirrorimage (L-nucleotide) of the naturally occurring (D-nucleotide), alsoreferred to as L-RNA in the case of a mirror ribonucleotide, and“spiegelmer”. The nucleotide can be a ribonucleotide or adeoxyribonucleotide and may further comprise at least one sugar, baseand or backbone modification. See U.S. Pat. No. 6,586,238. Also, U.S.Pat. No. 6,602,858 discloses nucleic acid catalysts comprising at leastone L-nucleotide substitution. Mirror nucleotide includes for exampleL-DNA (L-deoxyriboadenosine-3′-phosphate (mirror dA);L-deoxyribocytidine-3′-phosphate (mirror dC);L-deoxyriboguanosine-3′-phosphate (mirror dG);L-deoxyribothymidine-3′-phosphate (mirror image dT)) and L-RNA(L-riboadenosine-3′-phosphate (mirror rA); L-ribocytidine-3′-phosphate(mirror rC); L-riboguanosine-3′-phosphate (mirror rG);L-ribouracil-3′-phosphate (mirror dU)).

The term “capping moiety” as used herein includes abasic ribose moiety,abasic deoxyribose moiety, modifications to abasic ribose and abasicdeoxyribose moieties including 2′O alkyl modifications; inverted abasicribose and abasic deoxyribose moieties and modifications thereof;C6-imino-Pi; a mirror nucleotide including L-DNA and L-RNA; 5′O-Menucleotide; and nucleotide analogs including 4′,5′-methylene nucleotide;1-(β-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;alpha-nucleotide; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted abasic moiety; 1,4-butanediol phosphate;5′-amino; and bridging or non bridging methylphosphonate and 5′-mercaptomoieties.

Certain preferred capping moieties are abasic ribose or abasicdeoxyribose moieties; inverted abasic ribose or abasic deoxyribosemoieties; C6-amino-Pi; a mirror nucleotide including L-DNA and L-RNA.

A further end modification is a biotin group. Such biotin group maypreferably be attached to either the most 5′ or the most 3′ nucleotideof the first and/or second strand or to both ends. In a more preferredembodiment the biotin group is coupled to a polypeptide or a protein. Itis also within the scope of the present invention that the polypeptideor protein is attached through any of the other aforementioned endmodifications.

The various end modifications as disclosed herein are preferably locatedat the ribose moiety of a nucleotide of the nucleic acid according tothe present invention. More particularly, the end modification may beattached to or replace any of the OH-groups of the ribose moiety,including but not limited to the 2′OH, 3′OH and 5′OH position, providedthat the nucleotide thus modified is a terminal nucleotide. Invertedabasic or abasic are nucleotides, either deoxyribonucleotides orribonucleotides which do not have a nucleobase moiety (for example seeSternberger, et al., (2002). Antisense Nucleic Acid Drug Dev, 12,131-43).

Modified deoxyribonucleotide includes, for example 5′OMe DNA(5-methyl-deoxyriboguanosine-3′-phosphate) which may be useful as anucleotide in the 5′ terminal position (position number 1); PACE(deoxyriboadenine 3′ phosphonoacetate, deoxyribocytidine 3′phosphonoacetate, deoxyriboguanosine 3′phosphonoacetate,deoxyribothymidine 3′ phosphonoacetate.

Bridged nucleic acids include LNA (2′-O,4′-C-methylene bridged NucleicAcid adenosine 3′ monophosphate, 2′-O,4′-C-methylene bridged NucleicAcid 5-methyl-cytidine 3′ monophosphate, 2′-O,4′-C-methylene bridgedNucleic Acid guanosine 3′ monophosphate, 5-methyl-uridine (or thymidine)3′ monophosphate); and ENA (2′-O,4′-C-ethylene bridged nucleic acidadenosine 3′ monophosphate, 2′-O,4′-C-ethylene bridged nucleic acid5-methyl-cytidine 3′ monophosphate, 2′-O,4′-C-ethylene bridged nucleicacid guanosine 3′ monophosphate, 5-methyl-uridine (or thymidine) 3′monophosphate).

In certain embodiments the complementarity between said first strand andthe target nucleic acid is perfect. In some embodiments, the strands aresubstantially complementary, i.e. having one, two or up to threemismatches between said first strand and the target nucleic acid.Substantially complementary refers to complementarity of greater thanabout 84%, to another sequence. For example in a duplex regionconsisting of 19 base pairs one mismatch results in 94.7%complementarity, two mismatches results in about 89.5% complementarityand 3 mismatches results in about 84.2% complementarity, rendering theduplex region substantially complementary. Accordingly substantiallyidentical refers to identity of greater than about 84%, to anothersequence.

In some embodiments the first strand and the second strand of thecompound are linked by a loop structure, which is comprised of anon-nucleic acid polymer such as, inter alia, polyethylene glycol.Alternatively, the loop structure is comprised of a nucleic acid,including modified and non-modified ribonucleotides and modified andnon-modified deoxyribonucleotides.

In further embodiments, the 5′-terminus of the first strand of the siRNAis linked to the 3′-terminus of the second strand, or the 3′-terminus ofthe first strand is linked to the 5′-terminus of the second strand, saidlinkage being via a nucleic acid linker typically having a lengthbetween 2-100 nucleobases, preferably about 2 to about 30 nucleobases.

In preferred embodiments the methods of the invention employoligonucleotide compounds having alternating ribonucleotides modified inat least one of the antisense and the sense strands of the compound, for19 mer and 23 mer oligomers the ribonucleotides at the 5′ and 3′ terminiof the antisense strand are modified in their sugar residues, and theribonucleotides at the 5′ and 3′ termini of the sense strand areunmodified in their sugar residues. For 21 mer oligomers theribonucleotides at the 5′ and 3′ termini of the sense strand aremodified in their sugar residues, and the ribonucleotides at the 5′ and3′ termini of the antisense strand are unmodified in their sugarresidues, or may have an optional additional modification at the 3′terminus. As mentioned above, it is preferred that the middle nucleotideof the antisense strand is unmodified.

According to one preferred embodiment of the invention, the antisenseand the sense strands of the oligonucleotide/siRNA are phosphorylated atthe 3′-terminus and not at the 5′-terminus. According to anotherpreferred embodiment of the invention, the antisense and the sensestrands are non-phosphorylated. According to yet another preferredembodiment of the invention, the 5′ most ribonucleotide in the sensestrand is modified to abolish any possibility of in vivo5′-phosphorylation.

Any siRNA sequence can be prepared having any of themodifications/structures disclosed herein. The compound comprising acombination of sequence plus structure is useful in the treatment of theconditions disclosed herein.

Structural Motifs

In some embodiments of the present invention the oligonucleotideinhibitor is chemically modified siRNA according to one of the followingmodifications set forth in Structures (A)-(P) or as tandem siRNA orRNAstar.

In one aspect the present invention provides a compound set forth asStructure (A):

-   (A) 5′ (N)_(x)-Z 3′ (antisense strand)    -   3′ Z′-(N′)_(y) 5′ (sense strand)        wherein each of N and N′ is a nucleotide selected from an        unmodified ribonucleotide, a modified ribonucleotide, an        unmodified deoxyribonucleotide and a modified        deoxyribonucleotide;        wherein each of (N)_(x) and (N′)_(y) is an oligonucleotide in        which each consecutive N or N′ is joined to the next N or N′ by        a covalent bond;        wherein each of x and y is an integer between 18 and 40;        wherein each of Z and Z′ may be present or absent, but if        present is 1-5 consecutive nucleotides covalently attached at        the 3′ terminus of the strand in which it is present;        wherein the sequence of (N′)_(y) is a sequence substantially        complementary to (N)x; and        wherein the sequence of (N)_(x) comprises an antisense sequence        substantially complementary to from about 18 to about 40        consecutive ribonucleotides in an mRNA of a target gene        associated with acute kidney injury.

In certain embodiments the present invention provides a compound havingstructure (B)

-   (B) 5′ (N)_(x)-Z 3′ (antisense strand)    -   3′ Z′-(N′)_(y) 5′ (sense strand)        wherein each of (N)_(x) and (N′)_(y) is an oligomer in which        each consecutive N or N′ is an unmodified ribonucleotide or a        modified ribonucleotide joined to the next N or N′ by a covalent        bond;        wherein each of Z and Z′ may be present or absent, but if        present is 1-5 consecutive nucleotides covalently attached at        the 3′ terminus of the strand in which it is present; wherein        each of x and y=19, 21 or 23 and (N), and (N′)_(y) are fully        complementary        wherein alternating ribonucleotides in each of (N)_(x) and        (N′)_(y) are modified to result in a 2′-O-methyl modification in        the sugar residue of the ribonucleotides;        wherein the sequence of (N′)_(y) is a sequence substantially        complementary to (N)x; and wherein the sequence of (N)_(x)        comprises an antisense sequence substantially complementary to        the substantially complementary to from about 18 to about 40        consecutive ribonucleotides in an mRNA of a target gene        associated with acute kidney injury.

In some embodiments each of (N)_(x) and (N′)_(y) is independentlyphosphorylated or non-phosphorylated at the 3′ and 5′ termini.

In certain embodiments wherein each of x and y=19 or 23, each N at the5′ and 3′ termini of (N)_(x) is modified; and each N′ at the 5′ and 3′termini of (N′)_(y) is unmodified.

In certain embodiments wherein each of x and y=21, each N at the 5′ and3′ termini of (N)_(x) is unmodified; and each N′ at the 5′ and 3′termini of (N′)_(y) is modified.

In particular embodiments, when x and y=19, the siRNA is modified suchthat a 2′-O-methyl (2′-OMe) group is present on the first, third, fifth,seventh, ninth, eleventh, thirteenth, fifteenth, seventeenth andnineteenth nucleotide of the antisense strand (N)_(x), and whereby thevery same modification, i. e. a 2′-OMe group, is present at the second,fourth, sixth, eighth, tenth, twelfth, fourteenth, sixteenth andeighteenth nucleotide of the sense strand (N′)_(y). In variousembodiments these particular siRNA compounds are blunt ended at bothtermini.

In some embodiments, the present invention provides a compound havingStructure (C):

-   (C) 5′ (N)x-Z 3′ antisense strand    -   3′ Z′-(N′)y 5′ sense strand        wherein each of N and N′ is a nucleotide independently selected        from an unmodified ribonucleotide, a modified ribonucleotide, an        unmodified deoxyribonucleotide and a modified        deoxyribonucleotide;        wherein each of (N)x and (N′)y is an oligomer in which each        consecutive nucleotide is joined to the next nucleotide by a        covalent bond and each of x and y is an integer between 18 and        40;        wherein in (N)x the nucleotides are unmodified or (N)x comprises        alternating modified ribonucleotides and unmodified        ribonucleotides; each modified ribonucleotide being modified so        as to have a 2′-O-methyl on its sugar and the ribonucleotide        located at the middle position of (N)x being modified or        unmodified preferably unmodified;        wherein (N′)y comprises unmodified ribonucleotides further        comprising one modified nucleotide at a terminal or penultimate        position, wherein the modified nucleotide is selected from the        group consisting of a mirror nucleotide, a bicyclic nucleotide,        a 2′-sugar modified nucleotide, an altritol nucleotide, or a        nucleotide joined to an adjacent nucleotide by an        internucleotide linkage selected from a 2′-5′ phosphodiester        bond, a P-alkoxy linkage or a PACE linkage;        wherein if more than one nucleotide is modified in (N′)y, the        modified nucleotides may be consecutive;        wherein each of Z and Z′ may be present or absent, but if        present is 1-5 deoxyribonucleotides covalently attached at the        3′ terminus of any oligomer to which it is attached;        wherein the sequence of (N′)_(y) comprises a sequence        substantially complementary to (N)x; and wherein (N)_(x)        comprises an antisense sequence substantially complementary to        from about 18 to about 40 consecutive ribonucleotides in an mRNA        of a target gene associated with acute kidney injury.

In particular embodiments, x=y=19 and in (N)x each modifiedribonucleotide is modified so as to have a 2′-O-methyl on its sugar andthe ribonucleotide located at the middle of (N)x is unmodified.Accordingly, in a compound wherein x=19, (N)x comprises 2′-O-methylsugar modified ribonucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15,17 and 19. In other embodiments, (N)x comprises 2′O Me modifiedribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17 and 19 and mayfurther comprise at least one abasic or inverted abasic unconventionalmoiety for example in position 5. In other embodiments, (N)x comprises2′O Me modified ribonucleotides at positions 2, 4, 8, 11, 13, 15, 17 and19 and may further comprise at least one abasic or inverted abasicunconventional moiety for example in position 6. In other embodiments,(N)x comprises 2′O Me modified ribonucleotides at positions 2, 4, 6, 8,11, 13, 17 and 19 and may further comprise at least one abasic orinverted abasic unconventional moiety for example in position 15. Inother embodiments, (N)x comprises 2′O Me modified ribonucleotides atpositions 2, 4, 6, 8, 11, 13, 15, 17 and 19 and may further comprise atleast one abasic or inverted abasic unconventional moiety for example inposition 14. In other embodiments, (N)x comprises 2′O Me modifiedribonucleotides at positions 1, 2, 3, 7, 9, 11, 13, 15, 17 and 19 andmay further comprise at least one abasic or inverted abasicunconventional moiety for example in position 5. In other embodiments,(N)x comprises 2′O Me modified ribonucleotides at positions 1, 2, 3, 5,7, 9, 11, 13, 15, 17 and 19 and may further comprise at least one abasicor inverted abasic unconventional moiety for example in position 6. Inother embodiments, (N)x comprises 2′O Me modified ribonucleotides atpositions 1, 2, 3, 5, 7, 9, 11, 13, 17 and 19 and may further compriseat least one abasic or inverted abasic unconventional moiety for examplein position 15. In other embodiments, (N)x comprises 2′O Me modifiedribonucleotides at positions 1, 2, 3, 5, 7, 9, 11, 13, 15, 17 and 19 andmay further comprise at least one abasic or inverted abasicunconventional moiety for example in position 14. In other embodiments,(N)x comprises 2′OMe modified ribonucleotides at positions 2, 4, 6, 7,9, 11, 13, 15, 17 and 19 and may further comprise at least one abasic orinverted abasic unconventional moiety for example in position 5. Inother embodiments, (N)x comprises 2′OMe modified ribonucleotides atpositions 1, 2, 4, 6, 7, 9, 11, 13, 15, 17 and 19 and may furthercomprise at least one abasic or inverted abasic unconventional moietyfor example in position 5. In other embodiments, (N)x comprises 2′O Memodified ribonucleotides at positions 2, 4, 6, 8, 11, 13, 14, 16, 17 and19 and may further comprise at least one abasic or inverted abasicunconventional moiety for example in position 15. In other embodiments,(N)x comprises 2′OMe modified ribonucleotides at positions 1, 2, 3, 5,7, 9, 11, 13, 14, 16, 17 and 19 and may further comprise at least oneabasic or inverted abasic unconventional moiety for example in position15. In other embodiments, (N)x comprises 2′OMe modified ribonucleotidesat positions 2, 4, 6, 8, 11, 13, 15, 17 and 19 and may further compriseat least one abasic or inverted abasic unconventional moiety for examplein position 7. In other embodiments, (N)x comprises 2′OMe modifiedribonucleotides at positions 2, 4, 6, 11, 13, 15, 17 and 19 and mayfurther comprise at least one abasic or inverted abasic unconventionalmoiety for example in position 8.

In yet other embodiments (N)x comprises at least one nucleotide mismatchrelative to the one of the genes. In certain preferred embodiments, (N)xcomprises a single nucleotide mismatch on position 5, 6, or 14. In oneembodiment of Structure (C), at least two nucleotides at either or boththe 5′ and 3′ termini of (N′)y are joined by a 2′-5′ phosphodiesterbond. In certain preferred embodiments x=y=19 or x=y=23; in (N)x thenucleotides alternate between modified ribonucleotides and unmodifiedribonucleotides, each modified ribonucleotide being modified so as tohave a 2′-O-methyl on its sugar and the ribonucleotide located at themiddle of (N)x being unmodified; and three nucleotides at the 3′terminus of (N′)y are joined by two 2′-5′ phosphodiester bonds (setforth herein as Structure I). In other preferred embodiments, x=y=19 orx=y=23; in (N)x the nucleotides alternate between modifiedribonucleotides and unmodified ribonucleotides, each modifiedribonucleotide being modified so as to have a 2′-O-methyl on its sugarand the ribonucleotide located at the middle of (N)x being unmodified;and four consecutive nucleotides at the 5′ terminus of (N′)y are joinedby three 2′-5′ phosphodiester bonds. In a further embodiment, anadditional nucleotide located in the middle position of (N)y may bemodified with 2′-O-methyl on its sugar. In another preferred embodiment,in (N)x the nucleotides alternate between 2′-O-methyl modifiedribonucleotides and unmodified ribonucleotides, and in (N′)y fourconsecutive nucleotides at the 5′ terminus are joined by three 2′-5′phosphodiester bonds and the 5′ terminal nucleotide or two or threeconsecutive nucleotides at the 5′ terminus comprise 3′-O-methylmodifications.

In certain preferred embodiments of Structure C, x=y=19 and in (N′)y thenucleotide in at least one position comprises a mirror nucleotide, adeoxyribonucleotide and a nucleotide joined to an adjacent nucleotide bya 2′-5′ internucleotide bond;.

In certain preferred embodiments of Structure C, x=y=19 and (N′)ycomprises a mirror nucleotide. In various embodiments the mirrornucleotide is an L-DNA nucleotide. In certain embodiments the L-DNA isL-deoxyribocytidine. In some embodiments (N′)y comprises L-DNA atposition 18. In other embodiments (N′)y comprises L-DNA at positions 17and 18. In certain embodiments (N′)y comprises L-DNA substitutions atpositions 2 and at one or both of positions 17 and 18. In certainembodiments (N′)y further comprises a 5′ terminal cap nucleotide such as5′-O-methyl DNA or an abasic or inverted abasic moiety as an overhang.

In yet other embodiments (N′)y comprises a DNA at position 15 and L-DNAat one or both of positions 17 and 18. In that structure, position 2 mayfurther comprise an L-DNA or an abasic unconventional moiety.

Other embodiments of Structure C are envisaged wherein x=y=21 or whereinx=y=23; in these embodiments the modifications for (N′)y discussed aboveinstead of being on positions 15, 16, 17, 18 are on positions 17, 18,19, 20 for 21 mer and on positions 19, 20, 21, 22 for 23 mer; similarlythe modifications at one or both of positions 17 and 18 are on one orboth of positions 19 or 20 for the 21 mer and one or both of positions21 and 22 for the 23 mer. All modifications in the 19 mer are similarlyadjusted for the 21 and 23 mer.

According to various embodiments of Structure (C), in (N′)y 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides at the 3′terminus are linked by 2′-5′ internucleotide linkages. In one preferredembodiment, four consecutive nucleotides at the 3′ terminus of (N′)y arejoined by three 2′-5′ phosphodiester bonds, wherein one or more of the2′-5′ nucleotides which form the 2′-5′ phosphodiester bonds furthercomprises a 3′-O-methyl sugar modification. Preferably the 3′ terminalnucleotide of (N′)y comprises a 2′-O-methyl sugar modification. Incertain preferred embodiments of Structure C, x=y=19 and in (N′)y two ormore consecutive nucleotides at positions 15, 16, 17, 18 and 19 comprisea nucleotide joined to an adjacent nucleotide by a 2′-5′ internucleotidebond. In various embodiments the nucleotide forming the 2′-5′internucleotide bond comprises a 3′ deoxyribose nucleotide or a 3′methoxy nucleotide. In some embodiments the nucleotides at positions 17and 18 in (N′)y are joined by a 2′-5′ internucleotide bond. In otherembodiments the nucleotides at positions 16, 17, 18, 16-17, 17-18, or16-18 in (N′)y are joined by a 2′-5′ internucleotide bond.

In certain embodiments (N′)y comprises an L-DNA at position 2 and 2′-5′internucleotide bonds at positions 16-17, 17-18, or 16-18. In certainembodiments (N′)y comprises 2′-5′ internucleotide bonds at positions16-17, 17-18, or 16-18 and a 5′ terminal cap nucleotide.

According to various embodiments of Structure (C), in (N′)y 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive nucleotides at eitherterminus or 2-8 modified nucleotides at each of the 5′ and 3′ terminiare independently mirror nucleotides. In some embodiments the mirrornucleotide is an L-ribonucleotide. In other embodiments the mirrornucleotide is an L-deoxyribonucleotide. The mirror nucleotide mayfurther be modified at the sugar or base moiety or in an internucleotidelinkage.

In one preferred embodiment of Structure (C), the 3′ terminal nucleotideor two or three consecutive nucleotides at the 3′ terminus of (N′)y areL-deoxyribonucleotides. In other embodiments of Structure (C), in (N′)y2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotidesat either terminus or 2-8 modified nucleotides at each of the 5′ and 3′termini are independently 2′ sugar modified nucleotides. In someembodiments the 2′ sugar modification comprises the presence of anamino, a fluoro, an alkoxy or an alkyl moiety. In certain embodimentsthe 2′ sugar modification comprises a methoxy moiety (2′-OMe). In oneseries of preferred embodiments, three, four or five consecutivenucleotides at the 5′ terminus of (N′)y comprise the 2′-OMemodification. In another preferred embodiment, three consecutivenucleotides at the 3′ terminus of (N′)y comprise the 2′-O-methylmodification.

In some embodiments of Structure (C), in (N′)y 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 or 14 consecutive ribonucleotides at either or 2-8modified nucleotides at each of the 5′ and 3′ termini are independentlybicyclic nucleotide. In various embodiments the bicyclic nucleotide is alocked nucleic acid (LNA). A 2′-O, 4′-C-ethylene-bridged nucleic acid(ENA) is a species of LNA (see below).

In various embodiments (N′)y comprises modified nucleotides at the 5′terminus or at both the 3′ and 5′ termini.

In some embodiments of Structure (C), at least two nucleotides at eitheror both the 5′ and 3′ termini of (N′)y are joined by P-ethoxy backbonemodifications. In certain preferred embodiments x=y=19 or x=y=23; in(N)x the nucleotides alternate between modified ribonucleotides andunmodified ribonucleotides, each modified ribonucleotide being modifiedso as to have a 2′-O-methyl on its sugar and the ribonucleotide locatedat the middle position of (N)x being unmodified; and four consecutivenucleotides at the 3′ terminus or at the 5′ terminus of (N′)y are joinedby three P-ethoxy backbone modifications. In another preferredembodiment, three consecutive nucleotides at the 3′ terminus or at the5′ terminus of (N′)y are joined by two P-ethoxy backbone modifications.

In some embodiments of Structure (C), in (N′)y 2, 3, 4, 5, 6, 7 or 8,consecutive ribonucleotides at each of the 5′ and 3′ termini areindependently mirror nucleotides, nucleotides joined by 2′-5′phosphodiester bond, 2′ sugar modified nucleotides or bicyclicnucleotide. In one embodiment, the modification at the 5′ and 3′ terminiof (N′)y is identical. In one preferred embodiment, four consecutivenucleotides at the 5′ terminus of (N′)y are joined by three 2′-5′phosphodiester bonds and three consecutive nucleotides at the 3′terminus of (N′)y are joined by two 2′-5′ phosphodiester bonds. Inanother embodiment, the modification at the 5′ terminus of (N′)y isdifferent from the modification at the 3′ terminus of (N′)y. In onespecific embodiment, the modified nucleotides at the 5′ terminus of(N′)y are mirror nucleotides and the modified nucleotides at the 3′terminus of (N′)y are joined by 2′-5′ phosphodiester bond. In anotherspecific embodiment, three consecutive nucleotides at the 5′ terminus of(N′)y are LNA nucleotides and three consecutive nucleotides at the 3′terminus of (N′)y are joined by two 2′-5′ phosphodiester bonds. In (N)xthe nucleotides alternate between modified ribonucleotides andunmodified ribonucleotides, each modified ribonucleotide being modifiedso as to have a 2′-O-methyl on its sugar and the ribonucleotide locatedat the middle of (N)x being unmodified, or the ribonucleotides in (N)xbeing unmodified.

In another embodiment of Structure (C), the present invention provides acompound wherein x=y=19 or x=y=23; in (N)x the nucleotides alternatebetween modified ribonucleotides and unmodified ribonucleotides, eachmodified ribonucleotide being modified so as to have a 2′-O-methyl onits sugar and the ribonucleotide located at the middle of (N)x beingunmodified; three nucleotides at the 3′ terminus of (N′)y are joined bytwo 2′-5′ phosphodiester bonds and three nucleotides at the 5′ terminusof (N′)y are LNA such as ENA.

In another embodiment of Structure (C), five consecutive nucleotides atthe 5′ terminus of (N′)y comprise the 2′-O-methyl sugar modification andtwo consecutive nucleotides at the 3′ terminus of (N′)y are L-DNA.

In yet another embodiment, the present invention provides a compoundwherein x=y=19 or x=y=23; (N)x consists of unmodified ribonucleotides;three consecutive nucleotides at the 3′ terminus of (N′)y are joined bytwo 2′-5′ phosphodiester bonds and three consecutive nucleotides at the5′ terminus of (N′)y are LNA such as ENA.

According to other embodiments of Structure (C), in (N′)y the 5′ or 3′terminal nucleotide, or 2, 3, 4, 5 or 6 consecutive nucleotides ateither termini or 1-4 modified nucleotides at each of the 5′ and 3′termini are independently phosphonocarboxylate or phosphinocarboxylatenucleotides (PACE nucleotides). In some embodiments the PACE nucleotidesare deoxyribonucleotides. In some preferred embodiments in (N′)y, 1 or 2consecutive nucleotides at each of the 5′ and 3′ termini are PACEnucleotides.

In some embodiments, the present invention provides a compound havingStructure (D):

-   -   (D) 5′ (N)x-Z 3′ antisense strand        -   3′ Z′-(N′)y 5′ sense strand            wherein each of N and N′ is a nucleotide selected from an            unmodified ribonucleotide, a modified ribonucleotide, an            unmodified deoxyribonucleotide or a modified            deoxyribonucleotide;            wherein each of (N)x and (N′)y is an oligomer in which each            consecutive nucleotide is joined to the next nucleotide by a            covalent bond and each of x and y is an integer between 18            and 40;            wherein (N)x comprises unmodified ribonucleotides further            comprising one modified nucleotide at the 3′ terminal or            penultimate position, wherein the modified nucleotide is            selected from the group consisting of a bicyclic nucleotide,            a 2′ sugar modified nucleotide, a mirror nucleotide, an            altritol nucleotide, or a nucleotide joined to an adjacent            nucleotide by an internucleotide linkage selected from a            2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE            linkage;            wherein (N′)y comprises unmodified ribonucleotides further            comprising one modified nucleotide at the 5′ terminal or            penultimate position, wherein the modified nucleotide is            selected from the group consisting of a bicyclic nucleotide,            a 2′ sugar modified nucleotide, a mirror nucleotide, an            altritol nucleotide, or a nucleotide joined to an adjacent            nucleotide by an internucleotide linkage selected from a            2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE            linkage;            wherein in each of (N)x and (N′)y modified and unmodified            nucleotides are not alternating;            wherein each of Z and Z′ may be present or absent, but if            present is 1-5 deoxyribonucleotides covalently attached at            the 3′ terminus of any oligomer to which it is attached;            wherein the sequence of (N′)X is a sequence substantially            complementary to (N)x; and wherein (N)_(x) comprises an            antisense sequence substantially complementary to from about            18 to about 40 consecutive ribonucleotides in an mRNA of a            target gene associated with acute kidney injury.

In one embodiment of Structure (D), x=y=19 or x=y=23; (N)x comprisesunmodified ribonucleotides in which two consecutive nucleotides linkedby one 2′-5′ internucleotide linkage at the 3′ terminus; and (N′)ycomprises unmodified ribonucleotides in which two consecutivenucleotides linked by one 2′-5′ internucleotide linkage at the 5′terminus.

In some embodiments, x=y=19 or x=y=23; (N)x comprises unmodifiedribonucleotides in which three consecutive nucleotides at the 3′terminus are joined together by two 2′-5′ phosphodiester bonds; and(N′)y comprises unmodified ribonucleotides in which four consecutivenucleotides at the 5′ terminus are joined together by three 2′-5′phosphodiester bonds (set forth herein as Structure II).

According to various embodiments of Structure (D) 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive ribonucleotides starting at theultimate or penultimate position of the 3′ terminus of (N)x and 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides startingat the ultimate or penultimate position of the 5′ terminus of (N′)y arelinked by 2′-5′ internucleotide linkages.

According to one preferred embodiment of Structure (D), four consecutivenucleotides at the 5′ terminus of (N′)y are joined by three 2′-5′phosphodiester bonds and three consecutive nucleotides at the 3′terminus of (N′)x are joined by two 2′-5′ phosphodiester bonds. Threenucleotides at the 5′ terminus of (N′)y and two nucleotides at the 3′terminus of (N′)x may also comprise 3′-O-methyl modifications.

According to various embodiments of Structure (D), 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive nucleotides starting at the ultimateor penultimate position of the 3′ terminus of (N)x and 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides starting at theultimate or penultimate position of the 5′ terminus of (N′)y areindependently mirror nucleotides. In some embodiments the mirror is anL-ribonucleotide. In other embodiments the mirror nucleotide isL-deoxyribonucleotide.

In other embodiments of Structure (D), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 consecutive ribonucleotides starting at the ultimate orpenultimate position of the 3′ terminus of (N)x and 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive ribonucleotides starting at theultimate or penultimate position of the 5′ terminus of (N′)y areindependently 2′ sugar modified nucleotides. In some embodiments the 2′sugar modification comprises the presence of an amino, a fluoro, analkoxy or an alkyl moiety. In certain embodiments the 2′ sugarmodification comprises a methoxy moiety (2′-OMe).

In one preferred embodiment of Structure (D), five consecutivenucleotides at the 5′ terminus of (N′)y comprise a 2′OMe sugarmodification and five consecutive nucleotides at the 3′ terminus of(N′)x comprise the 2′OMe sugar modification. In another preferredembodiment of Structure (D), ten consecutive nucleotides at the 5′terminus of (N′)y comprise the 2′OMe sugar modification and fiveconsecutive nucleotides at the 3′ terminus of (N′)x comprise the 2′OMesugar modification. In another preferred embodiment of Structure (D),thirteen consecutive nucleotides at the 5′ terminus of (N′)y comprisethe 2′OMe sugar modification and five consecutive nucleotides at the 3′terminus of (N′)x comprise the 2′-O-methyl modification.

In some embodiments of Structure (D), in (N′)y 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 or 14 consecutive ribonucleotides starting at theultimate or penultimate position of the 3′ terminus of (N)x and 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides startingat the ultimate or penultimate position of the 5′ terminus of (N′)y areindependently a bicyclic nucleotide. In various embodiments the bicyclicnucleotide is a locked nucleic acid (LNA) such as a2′-O,4′-C-ethylene-bridged nucleic acid (ENA).

In various embodiments of Structure (D), (N′)y comprises a modifiednucleotide selected from a bicyclic nucleotide, a 2′ sugar modifiednucleotide, a mirror nucleotide, an altritol nucleotide or a nucleotidejoined to an adjacent nucleotide by an internucleotide linkage selectedfrom a 2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE linkage;

In various embodiments of Structure (D), (N)x comprises a modifiednucleotide selected from a bicyclic nucleotide, a 2′ sugar modifiednucleotide, a mirror nucleotide, an altritol nucleotide or a nucleotidejoined to an adjacent nucleotide by an internucleotide linkage selectedfrom a 2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE linkage;

In embodiments wherein each of the 3′ and 5′ termini of the same strandcomprises a modified nucleotide, the modification at the 5′ and 3′termini is identical. In another embodiment, the modification at the 5′terminus is different from the modification at the 3′ terminus of thesame strand. In one specific embodiment, the modified nucleotides at the5′ terminus are mirror nucleotides and the modified nucleotides at the3′ terminus of the same strand are joined by 2′-5′ phosphodiester bond.

In one specific embodiment of Structure (D), five consecutivenucleotides at the 5′ terminus of (N′)y comprise the 2′OMe sugarmodification and two consecutive nucleotides at the 3′ terminus of (N′)yare L-DNA. In addition, the compound may further comprise fiveconsecutive 2′OMe sugar modified nucleotides at the 3′ terminus of(N′)x.

In various embodiments of Structure (D), the modified nucleotides in(N)x are different from the modified nucleotides in (N′)y. For example,the modified nucleotides in (N)x are 2′ sugar modified nucleotides andthe modified nucleotides in (N′)y are nucleotides linked by 2′-5′internucleotide linkages. In another example, the modified nucleotidesin (N)x are mirror nucleotides and the modified nucleotides in (N′)y arenucleotides linked by 2′-5′ internucleotide linkages. In anotherexample, the modified nucleotides in (N)x are nucleotides linked by2′-5′ internucleotide linkages and the modified nucleotides in (N′)y aremirror nucleotides.

In additional embodiments, the present invention provides a compoundhaving Structure (E):

-   -   (E) 5′ (N)x-Z 3′ antisense strand        -   3′ Z′-(N′)y 5′ sense strand            wherein each of N and N′ is a nucleotide selected from an            unmodified ribonucleotide, a modified ribonucleotide, an            unmodified deoxyribonucleotide or a modified            deoxyribonucleotide;            wherein each of (N)x and (N′)y is an oligomer in which each            consecutive nucleotide is joined to the next nucleotide by a            covalent bond and each of x and y is an integer between 18            and 40;            wherein (N)x comprises unmodified ribonucleotides further            comprising one modified nucleotide at the 5′ terminal or            penultimate position, wherein the modified nucleotide is            selected from the group consisting of a bicyclic nucleotide,            a 2′ sugar modified nucleotide, a mirror nucleotide, an            altritol nucleotide, or a nucleotide joined to an adjacent            nucleotide by an internucleotide linkage selected from a            2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE            linkage;            wherein (N′)y comprises unmodified ribonucleotides further            comprising one modified nucleotide at the 3′ terminal or            penultimate position, wherein the modified nucleotide is            selected from the group consisting of a bicyclic nucleotide,            a 2′ sugar modified nucleotide, a mirror nucleotide, an            altritol nucleotide, or a nucleotide joined to an adjacent            nucleotide by an internucleotide linkage selected from a            2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE            linkage;            wherein in each of (N)x and (N′)y modified and unmodified            nucleotides are not alternating;            wherein each of Z and Z′ may be present or absent, but if            present is 1-5 deoxyribonucleotides covalently attached at            the 3′ terminus of any oligomer to which it is attached;            wherein the sequence of (N′)_(y) is a sequence substantially            complementary to (N)x; and wherein the sequence of (N)_(x)            comprises an antisense sequence substantially complementary            to from about 18 to about 40 consecutive ribonucleotides in            an mRNA of a target gene associated with acute kidney            injury.

In certain preferred embodiments the ultimate nucleotide at the 5′terminus of (N)x is unmodified.

According to various embodiments of Structure (E) 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive ribonucleotides starting at theultimate or penultimate position of the 5′ terminus of (N)x, preferablystarting at the 5′ penultimate position, and 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14 consecutive ribonucleotides starting at the ultimate orpenultimate position of the 3′ terminus of (N′)y are linked by 2′-5′internucleotide linkages.

According to various embodiments of Structure (E), 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive nucleotides starting at the ultimateor penultimate position of the 5′ terminus of (N)x, preferably startingat the 5′ penultimate position, and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13 or 14 consecutive nucleotides starting at the ultimate or penultimateposition of the 3′ terminus of (N′)y are independently mirrornucleotides. In some embodiments the mirror is an L-ribonucleotide. Inother embodiments the mirror nucleotide is L-deoxyribonucleotide.

In other embodiments of Structure (E), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 consecutive ribonucleotides starting at the ultimate orpenultimate position of the 5′ terminus of (N)x, preferably starting atthe 5′ penultimate position, and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13or 14 consecutive ribonucleotides starting at the ultimate orpenultimate position of the 3′ terminus of (N′)y are independently 2′sugar modified nucleotides. In some embodiments the 2′ sugarmodification comprises the presence of an amino, a fluoro, an alkoxy oran alkyl moiety. In certain embodiments the 2′ sugar modificationcomprises a methoxy moiety (2′-OMe).

In some embodiments of Structure (E), in (N′)y 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 or 14 consecutive ribonucleotides starting at theultimate or penultimate position of the 5′ terminus of (N)x, preferablystarting at the 5′ penultimate position, and 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14 consecutive ribonucleotides starting at the ultimate orpenultimate position of the 3′ terminus of (N′)y are independently abicyclic nucleotide. In various embodiments the bicyclic nucleotide is alocked nucleic acid (LNA) such as a 2′-O, 4′-C-ethylene-bridged nucleicacid (ENA). In various embodiments of Structure (E), (N′)y comprisesmodified nucleotides selected from a bicyclic nucleotide, a 2′ sugarmodified nucleotide, a mirror nucleotide, an altritol nucleotide, anucleotide joined to an adjacent nucleotide by a P-alkoxy backbonemodification or a nucleotide joined to an adjacent nucleotide by aninternucleotide linkage selected from a 2′-5′ phosphodiester bond, aP-alkoxy linkage or a PACE linkage at the 3′ terminus or at each of the3′ and 5′ termini.

In various embodiments of Structure (E), (N)x comprises a modifiednucleotide selected from a bicyclic nucleotide, a 2′ sugar modifiednucleotide, a mirror nucleotide, an altritol nucleotide, or a nucleotidejoined to an adjacent nucleotide by an internucleotide linkage selectedfrom a 2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE linkageat the 5′ terminus or at each of the 3′ and 5′ termini.

In one embodiment where both 3′ and 5′ termini of the same strandcomprise a modified nucleotide, the modification at the 5′ and 3′termini is identical. In another embodiment, the modification at the 5′terminus is different from the modification at the 3′ terminus of thesame strand. In one specific embodiment, the modified nucleotides at the5′ terminus are mirror nucleotides and the modified nucleotides at the3′ terminus of the same strand are joined by 2′-5′ phosphodiester bond.

In various embodiments of Structure (E), the modified nucleotides in(N)x are different from the modified nucleotides in (N′)y. For example,the modified nucleotides in (N)x are 2′ sugar modified nucleotides andthe modified nucleotides in (N′)y are nucleotides linked by 2′-5′internucleotide linkages. In another example, the modified nucleotidesin (N)x are mirror nucleotides and the modified nucleotides in (N′)y arenucleotides linked by 2′-5′ internucleotide linkages. In anotherexample, the modified nucleotides in (N)x are nucleotides linked by2′-5′ internucleotide linkages and the modified nucleotides in (N′)y aremirror nucleotides.

In additional embodiments, the present invention provides a compoundhaving Structure (F):

-   -   (F) 5′ (N)x-Z 3′ antisense strand        -   3′ Z′-(N′)y 5′ sense strand            wherein each of N and N′ is a nucleotide selected from an            unmodified ribonucleotide, a modified ribonucleotide, an            unmodified deoxyribonucleotide or a modified            deoxyribonucleotide;            wherein each of (N)x and (N′)y is an oligomer in which each            consecutive nucleotide is joined to the next nucleotide by a            covalent bond and each of x and y is an integer between 18            and 40;            wherein each of (N)x and (N′)y comprise unmodified            ribonucleotides in which each of (N)x and (N′)y            independently comprise one modified nucleotide at the 3′            terminal or penultimate position wherein the modified            nucleotide is selected from the group consisting of a            bicyclic nucleotide, a 2′ sugar modified nucleotide, a            mirror nucleotide, a nucleotide joined to an adjacent            nucleotide by a P-alkoxy backbone modification or a            nucleotide joined to an adjacent nucleotide by a 2′-5′            phosphodiester bond;            wherein in each of (N)x and (N′)y modified and unmodified            nucleotides are not alternating;            wherein each of Z and Z′ may be present or absent, but if            present is 1-5 deoxyribonucleotides covalently attached at            the 3′ terminus of any oligomer to which it is attached;            wherein the sequence of (N′)_(y) is a sequence substantially            complementary to (N)x; and wherein the sequence of (N)_(x)            comprises an antisense sequence substantially complementary            to from about 18 to about 40 consecutive ribonucleotides in            an mRNA of a target gene associated with acute kidney            injury.

In some embodiments of Structure (F), x=y=19 or x=y=23; (N′)y comprisesunmodified ribonucleotides in which two consecutive nucleotides at the3′ terminus comprises two consecutive mirror deoxyribonucleotides; and(N)x comprises unmodified ribonucleotides in which one nucleotide at the3′ terminus comprises a mirror deoxyribonucleotide (set forth asStructure III).

According to various embodiments of Structure (F) 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive ribonucleotides independentlybeginning at the ultimate or penultimate position of the 3′ termini of(N)x and (N′)y are linked by 2′-5′ internucleotide linkages.

According to one preferred embodiment of Structure (F), threeconsecutive nucleotides at the 3′ terminus of (N′)y are joined by two2′-5′ phosphodiester bonds and three consecutive nucleotides at the 3′terminus of (N′)x are joined by two 2′-5′ phosphodiester bonds.

According to various embodiments of Structure (F), 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive nucleotides independently beginningat the ultimate or penultimate position of the 3′ termini of (N)x and(N′)y are independently mirror nucleotides. In some embodiments themirror nucleotide is an L-ribonucleotide. In other embodiments themirror nucleotide is an L-deoxyribonucleotide.

In other embodiments of Structure (F), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 consecutive ribonucleotides independently beginning at theultimate or penultimate position of the 3′ termini of (N)x and (N′)y areindependently 2′ sugar modified nucleotides. In some embodiments the 2′sugar modification comprises the presence of an amino, a fluoro, analkoxy or an alkyl moiety. In certain embodiments the 2′ sugarmodification comprises a methoxy moiety (2′-OMe).

In some embodiments of Structure (F), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 consecutive ribonucleotides independently beginning at theultimate or penultimate position of the 3′ termini of (N)x and (N′)y areindependently a bicyclic nucleotide. In various embodiments the bicyclicnucleotide is a locked nucleic acid (LNA) such as a 2′-O,4′-C-ethylene-bridged nucleic acid (ENA).

In various embodiments of Structure (F), (N′)y comprises a modifiednucleotide selected from a bicyclic nucleotide, a 2′ sugar modifiednucleotide, a mirror nucleotide, an altritol nucleotide, or a nucleotidejoined to an adjacent nucleotide by an internucleotide linkage selectedfrom a 2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE linkageat the 3′ terminus or at both the 3′ and 5′ termini.

In various embodiments of Structure (F), (N)x comprises a modifiednucleotide selected from a bicyclic nucleotide, a 2′ sugar modifiednucleotide, a mirror nucleotide, an altritol nucleotide, or a nucleotidejoined to an adjacent nucleotide by an internucleotide linkage selectedfrom a 2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE linkageat the 3′ terminus or at each of the 3′ and 5′ termini.

In one embodiment where each of 3′ and 5′ termini of the same strandcomprise a modified nucleotide, the modification at the 5′ and 3′termini is identical. In another embodiment, the modification at the 5′terminus is different from the modification at the 3′ terminus of thesame strand. In one specific embodiment, the modified nucleotides at the5′ terminus are mirror nucleotides and the modified nucleotides at the3′ terminus of the same strand are joined by 2′-5′ phosphodiester bond.

In various embodiments of Structure (F), the modified nucleotides in(N)x are different from the modified nucleotides in (N′)y. For example,the modified nucleotides in (N)x are 2′ sugar modified nucleotides andthe modified nucleotides in (N′)y are nucleotides linked by 2′-5′internucleotide linkages. In another example, the modified nucleotidesin (N)x are mirror nucleotides and the modified nucleotides in (N′)y arenucleotides linked by 2′-5′ internucleotide linkages. In anotherexample, the modified nucleotides in (N)x are nucleotides linked by2′-5′ internucleotide linkages and the modified nucleotides in (N′)y aremirror nucleotides.

In additional embodiments, the present invention provides a compoundhaving Structure (G):

-   -   (G) 5′ (N)x-Z 3′ antisense strand        -   3′ Z′-(N′)y 5′ sense strand            wherein each of N and N′ is a nucleotide selected from an            unmodified ribonucleotide, a modified ribonucleotide, an            unmodified deoxyribonucleotide or a modified            deoxyribonucleotide;            wherein each of (N)x and (N′)y is an oligomer in which each            consecutive nucleotide is joined to the next nucleotide by a            covalent bond and each of x and y is an integer between 18            and 40;            wherein each of (N)x and (N′)y comprise unmodified            ribonucleotides in which each of (N)x and (N′)y            independently comprise one modified nucleotide at the 5′            terminal or penultimate position wherein the modified            nucleotide is selected from the group consisting of a            bicyclic nucleotide, a 2′ sugar modified nucleotide, a            mirror nucleotide, a nucleotide joined to an adjacent            nucleotide by a P-alkoxy backbone modification or a            nucleotide joined to an adjacent nucleotide by a 2′-5′            phosphodiester bond;            wherein for (N)x the modified nucleotide is preferably at            penultimate position of the 5′ terminal;            wherein in each of (N)x and (N′)y modified and unmodified            nucleotides are not alternating;            wherein each of Z and Z′ may be present or absent, but if            present is 1-5 deoxyribonucleotides covalently attached at            the 3′ terminus of any oligomer to which it is attached;            wherein the sequence of (N′)_(y) is a sequence substantially            complementary to (N)x; and wherein the sequence of (N)x            comprises an antisense sequence substantially complementary            to from about 18 to about 40 consecutive ribonucleotides in            an mRNA of a target gene associated with acute kidney            injury.

In some embodiments of Structure (G), x=y=19 or x=y=23.

According to various embodiments of Structure (G) 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive ribonucleotides independentlybeginning at the ultimate or penultimate position of the 5′ termini of(N)x and (N′)y are linked by 2′-5′ internucleotide linkages. For (N)xthe modified nucleotides preferably starting at the penultimate positionof the 5′ terminal.

According to various embodiments of Structure (G), 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive nucleotides independently beginningat the ultimate or penultimate position of the 5′ termini of (N)x and(N′)y are independently mirror nucleotides. In some embodiments themirror nucleotide is an L-ribonucleotide. In other embodiments themirror nucleotide is an L-deoxyribonucleotide. For (N)x the modifiednucleotides preferably starting at the penultimate position of the 5′terminal.

In other embodiments of Structure (G), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 consecutive ribonucleotides independently beginning at theultimate or penultimate position of the 5′ termini of (N)x and (N′)y areindependently 2′ sugar modified nucleotides. In some embodiments the 2′sugar modification comprises the presence of an amino, a fluoro, analkoxy or an alkyl moiety. In certain embodiments the 2′ sugarmodification comprises a methoxy moiety (2′-OMe). In some preferredembodiments the consecutive modified nucleotides preferably begin at thepenultimate position of the 5′ terminus of (N)x.

In one preferred embodiment of Structure (G), five consecutiveribonucleotides at the 5′ terminus of (N′)y comprise a 2′OMe sugarmodification and one ribonucleotide at the 5′ penultimate position of(N′)x comprises a 2′OMe sugar modification. In another preferredembodiment of Structure (G), five consecutive ribonucleotides at the 5′terminus of (N′)y comprise 2′OMe sugar modification and two consecutiveribonucleotides at the 5′ terminal position of (N′)x comprise a 2′OMesugar modification.

In some embodiments of Structure (G), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 consecutive ribonucleotides independently beginning at theultimate or penultimate position of the 5′ termini of (N)x and (N′)y arebicyclic nucleotides. In various embodiments the bicyclic nucleotide isa locked nucleic acid (LNA) such as a 2′-O, 4′-C-ethylene-bridgednucleic acid (ENA). In some preferred embodiments the consecutivemodified nucleotides preferably begin at the penultimate position of the5′ terminus of (N)x.

In various embodiments of Structure (G), (N′)y comprises a modifiednucleotide selected from a bicyclic nucleotide, a 2′ sugar modifiednucleotide, a mirror nucleotide, an altritol nucleotide, or a nucleotidejoined to an adjacent nucleotide by an internucleotide linkage selectedfrom a 2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE linkageat the 5′ terminus or at each of the 3′ and 5′ termini.

In various embodiments of Structure (G), (N)x comprises a modifiednucleotide selected from a bicyclic nucleotide, a 2′ sugar modifiednucleotide, a mirror nucleotide, an altritol nucleotide, or a nucleotidejoined to an adjacent nucleotide by an internucleotide linkage selectedfrom a 2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE linkageat the 5′ terminus or at each of the 3′ and 5′ termini.

In one embodiment where each of 3′ and 5′ termini of the same strandcomprise a modified nucleotide, the modification at the 5′ and 3′termini is identical. In another embodiment, the modification at the 5′terminus is different from the modification at the 3′ terminus of thesame strand. In one specific embodiment, the modified nucleotides at the5′ terminus are mirror nucleotides and the modified nucleotides at the3′ terminus of the same strand are joined by 2′-5′ phosphodiester bond.In various embodiments of Structure (G), the modified nucleotides in(N)x are different from the modified nucleotides in (N′)y. For example,the modified nucleotides in (N)x are 2′ sugar modified nucleotides andthe modified nucleotides in (N′)y are nucleotides linked by 2′-5′internucleotide linkages. In another example, the modified nucleotidesin (N)x are mirror nucleotides and the modified nucleotides in (N′)y arenucleotides linked by 2′-5′ internucleotide linkages. In anotherexample, the modified nucleotides in (N)x are nucleotides linked by2′-5′ internucleotide linkages and the modified nucleotides in (N′)y aremirror nucleotides.

In additional embodiments, the present invention provides a compoundhaving Structure (H):

-   -   (H) 5′ (N)x-Z 3′ antisense strand        -   3′ Z′-(N′)y 5′ sense strand            wherein each of N and N′ is a nucleotide selected from an            unmodified ribonucleotide, a modified ribonucleotide, an            unmodified deoxyribonucleotide or a modified            deoxyribonucleotide;            wherein each of (N)x and (N′)y is an oligomer in which each            consecutive nucleotide is joined to the next nucleotide by a            covalent bond and each of x and y is an integer between 18            and 40;            wherein (N)x comprises unmodified ribonucleotides further            comprising one modified nucleotide at the 3′ terminal or            penultimate position or the 5′ terminal or penultimate            position, wherein the modified nucleotide is selected from            the group consisting of a bicyclic nucleotide, a 2′ sugar            modified nucleotide, a mirror nucleotide, an altritol            nucleotide, or a nucleotide joined to an adjacent nucleotide            by an internucleotide linkage selected from a 2′-5′            phosphodiester bond, a P-alkoxy linkage or a PACE linkage;            wherein (N′)y comprises unmodified ribonucleotides further            comprising one modified nucleotide at an internal position,            wherein the modified nucleotide is selected from the group            consisting of a bicyclic nucleotide, a 2′ sugar modified            nucleotide, a mirror nucleotide, an altritol nucleotide, or            a nucleotide joined to an adjacent nucleotide by an            internucleotide linkage selected from a 2′-5′ phosphodiester            bond, a P-alkoxy linkage or a PACE linkage;            wherein in each of (N)x and (N′)y modified and unmodified            nucleotides are not alternating;            wherein each of Z and Z′ may be present or absent, but if            present is 1-5 deoxyribonucleotides covalently attached at            the 3′ terminus of any oligomer to which it is attached;            wherein the sequence of (N′)_(y) is a sequence substantially            complementary to (N)x; and wherein the sequence of (N)_(x)            comprises an antisense sequence substantially complementary            to from about 18 to about 40 consecutive ribonucleotides in            an mRNA of a target gene associated with acute kidney            injury.

In one embodiment of Structure (H), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13 or 14 consecutive ribonucleotides independently beginning at theultimate or penultimate position of the 3′ terminus or the 5′ terminusor both termini of (N)x are independently 2′ sugar modified nucleotides,bicyclic nucleotides, mirror nucleotides, altritol nucleotides ornucleotides joined to an adjacent nucleotide by a 2′-5′ phosphodiesterbond and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutiveinternal ribonucleotides in (N′)y are independently 2′ sugar modifiednucleotides, bicyclic nucleotides, mirror nucleotides, altritolnucleotides or nucleotides joined to an adjacent nucleotide by a 2′-5′phosphodiester bond. In some embodiments the 2′ sugar modificationcomprises the presence of an amino, a fluoro, an alkoxy or an alkylmoiety. In certain embodiments the 2′ sugar modification comprises amethoxy moiety (2′-OMe).

In another embodiment of Structure (H), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 consecutive ribonucleotides independently beginning at theultimate or penultimate position of the 3′ terminus or the 5′ terminusor 2-8 consecutive nucleotides at each of 5′ and 3′ termini of (N′)y areindependently 2′ sugar modified nucleotides, bicyclic nucleotides,mirror nucleotides, altritol nucleotides or nucleotides joined to anadjacent nucleotide by a 2′-5′ phosphodiester bond, and 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13 or 14 consecutive internal ribonucleotides in(N)x are independently 2′ sugar modified nucleotides, bicyclicnucleotides, mirror nucleotides, altritol nucleotides or nucleotidesjoined to an adjacent nucleotide by a 2′-5′ phosphodiester bond.

In one embodiment wherein each of 3′ and 5′ termini of the same strandcomprises a modified nucleotide, the modification at the 5′ and 3′termini is identical. In another embodiment, the modification at the 5′terminus is different from the modification at the 3′ terminus of thesame strand. In one specific embodiment, the modified nucleotides at the5′ terminus are mirror nucleotides and the modified nucleotides at the3′ terminus of the same strand are joined by 2′-5′ phosphodiester bond.

In various embodiments of Structure (H), the modified nucleotides in(N)x are different from the modified nucleotides in (N′)y. For example,the modified nucleotides in (N)x are 2′ sugar modified nucleotides andthe modified nucleotides in (N′)y are nucleotides linked by 2′-5′internucleotide linkages. In another example, the modified nucleotidesin (N)x are mirror nucleotides and the modified nucleotides in (N′)y arenucleotides linked by 2′-5′ internucleotide linkages. In anotherexample, the modified nucleotides in (N)x are nucleotides linked by2′-5′ internucleotide linkages and the modified nucleotides in (N′)y aremirror nucleotides.

In one preferred embodiment of Structure (H), x=y=19; three consecutiveribonucleotides at the 9-11 nucleotide positions 9-11 of (N′)y comprise2′OMe sugar modification and five consecutive ribonucleotides at the 3′terminal position of (N′)x comprise 2′OMe sugar modification.

For all the above Structures (A)-(H), in various embodiments x=y andeach of x and y is 19, 20, 21, 22 or 23. In certain embodiments, x=y=19.In yet other embodiments x=y=23. In additional embodiments the compoundcomprises modified ribonucleotides in alternating positions wherein eachN at the 5′ and 3′ termini of (N)x are modified in their sugar residuesand the middle ribonucleotide is not modified, e.g. ribonucleotide inposition 10 in a 19-mer strand, position 11 in a 21 mer and position 12in a 23-mer strand.

In some embodiments where x=y=21 or x=y=23 the position of modificationsin the 19 mer are adjusted for the 21 and 23 mers with the proviso thatthe middle nucleotide of the antisense strand is preferably notmodified.

In some embodiments, neither (N)x nor (N′)y are phosphorylated at the 3′and 5′ termini. In other embodiments either or both (N)x and (N′)y arephosphorylated at the 3′ termini. In yet another embodiment, either orboth (N)x and (N′)y are phosphorylated at the 3′ termini usingnon-cleavable phosphate groups. In yet another embodiment, either orboth (N)x and (N′)y are phosphorylated at the terminal 2′ terminiposition using cleavable or non-cleavable phosphate groups. Theseparticular siRNA compounds are also blunt ended and arenon-phosphorylated at the termini; however, comparative experiments haveshown that siRNA compounds phosphorylated at one or both of the3′-termini have similar activity in vivo compared to thenon-phosphorylated compounds.

In certain embodiments for all the above-mentioned Structures, thecompound is blunt ended, for example wherein both Z and Z′ are absent.In an alternative embodiment, the compound comprises at least one 3′overhang, wherein at least one of Z or Z′ is present. Z and Z′independently comprises one or more covalently linked modified ornon-modified nucleotides, for example inverted dT or dA; dT, LNA, mirrornucleotide and the like. In some embodiments each of Z and Z′ areindependently selected from dT and dTdT. siRNA in which Z and/or Z′ ispresent have similar activity and stability as siRNA in which Z and Z′are absent.

In certain embodiments for all the above-mentioned Structures, thecompound comprises one or more phosphonocarboxylate and/orphosphinocarboxylate nucleotides (PACE nucleotides). In some embodimentsthe PACE nucleotides are deoxyribonucleotides and thephosphinocarboxylate nucleotides are phosphinoacetate nucleotides.Examples of PACE nucleotides and analogs are disclosed in U.S. Pat. Nos.6,693,187 and 7,067,641, both incorporated herein by reference.

In certain embodiments for all the above-mentioned Structures, thecompound comprises one or more locked nucleic acids (LNA) also definedas bridged nucleic acids or bicyclic nucleotides. Preferred lockednucleic acids are 2′-O, 4′-C-ethylene nucleosides (ENA) or 2′-O,4′-C-methylene nucleosides. Other examples of LNA and ENA nucleotidesare disclosed in WO 98/39352, WO 00/47599 and WO 99/14226, allincorporated herein by reference.

In certain embodiments for all the above-mentioned Structures, thecompound comprises one or more altritol monomers (nucleotides), alsodefined as 1,5 anhydro-2-deoxy-D-altrito-hexitol (see for example,Allart, et al., 1998. Nucleosides & Nucleotides 17:1523-1526; Herdewijnet al., 1999. Nucleosides & Nucleotides 18:1371-1376; Fisher et al.,2007, NAR 35(4):1064-1074; all incorporated herein by reference).

The present invention explicitly excludes compounds in which each of Nand/or N′ is a deoxyribonucleotide (D-A, D-C, D-G, D-T). In certainembodiments (N)x and (N′)y may comprise independently 1, 2, 3, 4, 5, 6,7, 8, 9 or more deoxyribonucleotides. In certain embodiments the presentinvention provides a compound wherein each of N is an unmodifiedribonucleotide and the 3′ terminal nucleotide or 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 or 14 consecutive nucleotides at the 3′ terminus of (N′)yare deoxyribonucleotides. In yet other embodiments each of N is anunmodified ribonucleotide and the 5′ terminal nucleotide or 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive nucleotides at the 5′terminus of (N′)y are deoxyribonucleotides. In further embodiments the5′ terminal nucleotide or 2, 3, 4, 5, 6, 7, 8, or 9 consecutivenucleotides at the 5′ terminus and 1, 2, 3, 4, 5, or 6 consecutivenucleotides at the 3′ termini of (N)x are deoxyribonucleotides and eachof N′ is an unmodified ribonucleotide. In yet further embodiments (N)xcomprises unmodified ribonucleotides and 1 or 2, 3 or 4 consecutivedeoxyribonucleotides independently at each of the 5′ and 3′ termini and1 or 2, 3, 4, 5 or 6 consecutive deoxyribonucleotides in internalpositions; and each of N′ is an unmodified ribonucleotide. In certainembodiments the 3′ terminal nucleotide or 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12 13 or 14 consecutive nucleotides at the 3′ terminus of (N′)y andthe terminal 5′ nucleotide or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 13 or14 consecutive nucleotides at the 5′ terminus of (N)x aredeoxyribonucleotides. The present invention excludes compounds in whicheach of N and/or N′ is a deoxyribonucleotide. In some embodiments the 5′terminal nucleotide of N or 2 or 3 consecutive of N and 1, 2, or 3 of N′is a deoxyribonucleotide. Certain examples of active DNA/RNA siRNAchimeras are disclosed in US patent publication 2005/0004064, andUi-Tei, 2008 (NAR 36(7):2136-2151) incorporated herein by reference intheir entirety.

Unless otherwise indicated, in preferred embodiments of the structuresdiscussed herein the covalent bond between each consecutive N or N′ is aphosphodiester bond.

An additional novel molecule provided by the present invention is anoligonucleotide comprising consecutive nucleotides wherein a firstsegment of such nucleotides encode a first inhibitory RNA molecule, asecond segment of such nucleotides encode a second inhibitory RNAmolecule, and a third segment of such nucleotides encode a thirdinhibitory RNA molecule. Each of the first, the second and the thirdsegment may comprise one strand of a double stranded RNA and the first,second and third segments may be joined together by a linker. Further,the oligonucleotide may comprise three double stranded segments joinedtogether by one or more linker.

Thus, one molecule provided by the present invention is anoligonucleotide comprising consecutive nucleotides which encode threeinhibitory RNA molecules; said oligonucleotide may possess a triplestranded structure, such that three double stranded arms are linkedtogether by one or more linker, such as any of the linkers presentedhereinabove. This molecule forms a “star”-like structure, and may alsobe referred to herein as RNAstar. Such structures are disclosed in PCTpatent publication WO 2007/091269, assigned to the assignee of thepresent invention and incorporated herein in its entirety by reference.

A covalent bond refers to an internucleotide linkage linking onenucleotide monomer to an adjacent nucleotide monomer. A covalent bondincludes for example, a phosphodiester bond, a phosphorothioate bond, aP-alkoxy bond, a P-carboxy bond and the like. The normal internucleosidelinkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. In certainpreferred embodiments a covalent bond is a phosphodiester bond. Covalentbond encompasses non-phosphorous-containing internucleoside linkages,such as those disclosed in WO 2004/041924 inter alia. Unless otherwiseindicated, in preferred embodiments of the structures discussed hereinthe covalent bond between each consecutive N or N′ is a phosphodiesterbond.

For all of the structures above, in some embodiments the oligonucleotidesequence of (N)x is fully complementary to the oligonucleotide sequenceof (N′)y. In other embodiments (N)x and (N′)y are substantiallycomplementary. In certain embodiments (N)x is fully complementary to atarget sequence. In other embodiments (N)x is substantiallycomplementary to a target sequence.

In some embodiments, neither (N)x nor (N′)y are phosphorylated at the 3′and 5′ termini. In other embodiments either or both (N)x and (N′)y arephosphorylated at the 3′ termini (3′ Pi). In yet another embodiment,either or both (N)x and (N′)y are phosphorylated at the 3′ termini withnon-cleavable phosphate groups. In yet another embodiment, either orboth (N)x and (N′)y are phosphorylated at the terminal 2′ terminiposition using cleavable or non-cleavable phosphate groups. Further, theinhibitory nucleic acid molecules of the present invention may compriseone or more gaps and/or one or more nicks and/or one or more mismatches.Without wishing to be bound by theory, gaps, nicks and mismatches havethe advantage of partially destabilizing the nucleic acid/siRNA, so thatit may be more easily processed by endogenous cellular machinery such asDICER, DROSHA or RISC into its inhibitory components.

In the context of the present invention, a gap in a nucleic acid refersto the absence of one or more internal nucleotides in one strand, whilea nick in a nucleic acid refers to the absence of an internucleotidelinkage between two adjacent nucleotides in one strand. Any of themolecules of the present invention may contain one or more gaps and/orone or more nicks.

In one aspect the present invention provides a compound having Structure(I):

-   -   (I) 5′ (N)x-Z 3′ (antisense strand)        -   3′ Z′-(N′)y-z″ 5′ (sense strand)            wherein each of N and N′ is a ribonucleotide which may be            unmodified or modified, or an unconventional moiety;            wherein each of (N)x and (N′)y is an oligonucleotide in            which each consecutive N or N′ is joined to the next N or N′            by a covalent bond;            wherein Z and Z′ may be present or absent, but if present is            independently 1-5 consecutive nucleotides or non-nucleotide            moieties covalently attached at the 3′ terminus of the            strand in which it is present;            wherein z″ may be present or absent, but if present is a            capping moiety covalently attached at the 5′ terminus of            (N′)y;            wherein x=18 to 27;            wherein y=18 to 27;            wherein (N)x comprises modified and unmodified            ribonucleotides, each modified ribonucleotide having a            2′-O-methyl on its sugar, wherein N at the 3′ terminus of            (N)x is a modified ribonucleotide, (N)x comprises at least            five alternating modified ribonucleotides beginning at the            3′ end and at least nine modified ribonucleotides in total            and each remaining N is an unmodified ribonucleotide;            wherein in (N′)y at least one unconventional moiety is            present, which unconventional moiety may be an abasic ribose            moiety, an abasic deoxyribose moiety, a modified or            unmodified deoxyribonucleotide, a mirror nucleotide, and a            nucleotide joined to an adjacent nucleotide by a 2′-5′            internucleotide phosphate bond; and            wherein the sequence of (N′)_(y) is a sequence having            complementarity to (N)x; and wherein the sequence of (N)_(x)            comprises an antisense sequence having complementarity to            from about 18 to about 27 consecutive ribonucleotides in an            mRNA of a target gene associated with acute kidney injury.

In some embodiments x=y=19. In other embodiments x=y=23. In someembodiments the at least one unconventional moiety is present atpositions 15, 16, 17, or 18 in (N′)y. In some embodiments theunconventional moiety is selected from a mirror nucleotide, an abasicribose moiety and an abasic deoxyribose moiety. In some preferredembodiments the unconventional moiety is a mirror nucleotide, preferablyan L-DNA moiety. In some embodiments an L-DNA moiety is present atposition 17, position 18 or positions 17 and 18.

In other embodiments the unconventional moiety is an abasic moiety. Invarious embodiments (N′)y comprises at least five abasic ribose moietiesor abasic deoxyribose moieties.

In yet other embodiments (N′)y comprises at least five abasic ribosemoieties or abasic deoxyribose moieties and at least one of N′ is anLNA.

In some embodiments (N)x comprises nine alternating modifiedribonucleotides. In other embodiments of Structure (I) (N)x comprisesnine alternating modified ribonucleotides further comprising a 2′Omodified nucleotide at position 2. In some embodiments (N)x comprises2′O Me modified ribonucleotides at the odd numbered positions 1, 3, 5,7, 9, 11, 13, 15, 17, 19. In other embodiments (N)x further comprises a2′O Me modified ribonucleotide at one or both of positions 2 and 18. Inyet other embodiments (N)x comprises 2′O Me modified ribonucleotides atpositions 2, 4, 6, 8, 11, 13, 15, 17, 19.

In various embodiments z″ is present and is selected from an abasicribose moiety, a deoxyribose moiety; an inverted abasic ribose moiety, adeoxyribose moiety; C6-amino-Pi; a mirror nucleotide.

In another aspect the present invention provides a compound havingStructure (J) set forth below:

-   -   (J) 5′ (N)x-Z 3′ (antisense strand)        -   3′ Z′-(N′)y-z″ 5′ (sense strand)            wherein each of N and N′ is a ribonucleotide which may be            unmodified or modified, or an unconventional moiety;            wherein each of (N)x and (N′)y is an oligonucleotide in            which each consecutive N or N′ is joined to the next N or N′            by a covalent bond;            wherein Z and Z′ may be present or absent, but if present is            independently 1-5 consecutive nucleotides covalently            attached at the 3′ terminus of the strand in which it is            present;            wherein z″ may be present or absent but if present is a            capping moiety covalently attached at the 5′ terminus of            (N′)y;            wherein x=18 to 27;            wherein y=18 to 27;            wherein (N)x comprises modified or unmodified            ribonucleotides, and optionally at least one unconventional            moiety;            wherein in (N′)y at least one unconventional moiety is            present, which unconventional moiety may be an abasic ribose            moiety, an abasic deoxyribose moiety, a modified or            unmodified deoxyribonucleotide, a mirror nucleotide, a            non-base pairing nucleotide analog or a nucleotide joined to            an adjacent nucleotide by a 2′-5′ internucleotide phosphate            bond; and            wherein the sequence of (N′)_(y) is a sequence having            complementarity to (N)x; and wherein the sequence of (N)_(x)            comprises an antisense sequence having complementarity to            from about 18 to about 27 consecutive ribonucleotides in an            mRNA of a target gene associated with acute kidney injury.

In some embodiments x=y=19. In other embodiments x=y=23. In somepreferred embodiments (N)x comprises modified and unmodifiedribonucleotides, and at least one unconventional moiety.

In some embodiments in (N)x the N at the 3′ terminus is a modifiedribonucleotide and (N)x comprises at least 8 modified ribonucleotides.In other embodiments at least 5 of the at least 8 modifiedribonucleotides are alternating beginning at the 3′ end. In someembodiments (N)x comprises an abasic moiety in one of positions 5, 6, 7,8, 9, 10, 11, 12, 13, 14 or 15.

In some embodiments the at least one unconventional moiety in (N′)y ispresent at positions 15, 16, 17, or 18. In some embodiments theunconventional moiety is selected from a mirror nucleotide, an abasicribose moiety and an abasic deoxyribose moiety. In some preferredembodiments the unconventional moiety is a mirror nucleotide, preferablyan L-DNA moiety. In some embodiments an L-DNA moiety is present atposition 17, position 18 or positions 17 and 18. In other embodimentsthe at least one unconventional moiety in (N′)y is an abasic ribosemoiety or an abasic deoxyribose moiety.

In yet another aspect the present invention provides a compound havingStructure (K) set forth below:

-   -   (K) 5′ (N)_(x)-Z 3′ (antisense strand)        -   3′ Z′-(N′)_(y)-z″ 5′ (sense strand)            wherein each of N and N′ is a ribonucleotide which may be            unmodified or modified, or an unconventional moiety;            wherein each of (N)x and (N′)y is an oligonucleotide in            which each consecutive N or N′ is joined to the next N or N′            by a covalent bond;            wherein Z and Z′ may be present or absent, but if present is            independently 1-5 consecutive nucleotides covalently            attached at the 3′ terminus of the strand in which it is            present;            wherein z″ may be present or absent but if present is a            capping moiety covalently attached at the 5′ terminus of            (N′)y;            wherein x=18 to 27;            wherein y=18 to 27;            wherein (N)x comprises a combination of modified or            unmodified ribonucleotides and unconventional moieties, any            modified ribonucleotide having a 2′-O-methyl on its sugar;            wherein (N′)y comprises modified or unmodified            ribonucleotides and optionally an unconventional moiety, any            modified ribonucleotide having a 2′OMe on its sugar, wherein            the sequence of (N′)_(y) is a sequence having            complementarity to (N)x; and wherein the sequence of (N)_(x)            comprises an antisense sequence having complementarity to            from about 18 to about 27 consecutive ribonucleotides in an            mRNA of a target gene associated with acute kidney injury.

In some embodiments x=y=19. In other embodiments x=y=23. In somepreferred embodiments the at least one unconventional moiety is presentin (N)x and is an abasic ribose moiety or an abasic deoxyribose moiety.In other embodiments the at least one unconventional moiety is presentin (N)x and is a non-base pairing nucleotide analog. In variousembodiments (N′)y comprises unmodified ribonucleotides. In someembodiments (N)x comprises at least five abasic ribose moieties orabasic deoxyribose moieties or a combination thereof. In certainembodiments (N)x and/or (N′)y comprise modified ribonucleotides which donot base pair with corresponding modified or unmodified ribonucleotidesin (N′)y and/or (N)x.

In various embodiments the present invention provides an siRNA set forthin Structure (L):

-   -   (L) 5′ (N)_(x)-Z 3′ (antisense strand)        -   3′ Z′-(N′)_(y) 5′ (sense strand)            wherein each of N and N′ is a nucleotide selected from an            unmodified ribonucleotide, a modified ribonucleotide, an            unmodified deoxyribonucleotide and a modified            deoxyribonucleotide;            wherein each of (N)_(x) and (N′)_(y) is an oligonucleotide            in which each consecutive N or N′ is joined to the next N or            N′ by a covalent bond;            wherein Z and Z′ are absent;            wherein x=y=19;            wherein in (N′)y the nucleotide in at least one of positions            15, 16, 17, 18 and 19 comprises a nucleotide selected from            an abasic unconventional moiety, a mirror nucleotide, a            deoxyribonucleotide and a nucleotide joined to an adjacent            nucleotide by a 2′-5′ internucleotide bond;            wherein (N)x comprises alternating 2′OMe sugar modified            ribonucleotides and unmodified ribonucleotides so as to have            2′OMe sugar modified ribonucleotide at the middle position            of (N)x; and            wherein the sequence of (N′)_(y) is a sequence having            complementarity to (N)x; and wherein the sequence of (N)_(x)            comprises an antisense having complementarity to from about            18 to about 40 consecutive ribonucleotides in an mRNA of a            target gene associated with acute kidney injury.

In some embodiments of Structure (L), in (N′)y the nucleotide in one orboth of positions 17 and 18 comprises a modified nucleotide selectedfrom an abasic unconventional moiety, a mirror nucleotide and anucleotide joined to an adjacent nucleotide by a 2′-5′ internucleotidebond. In some embodiments the mirror nucleotide is selected from L-DNAand L-RNA. In various embodiments the mirror nucleotide is L-DNA.

In various embodiments (N′)y comprises a modified nucleotide at position15 wherein the modified nucleotide is selected from a mirror nucleotideand a deoxyribonucleotide.

In certain embodiments (N′)y further comprises a modified nucleotide orpseudo nucleotide at position 2 wherein the pseudo nucleotide may be anabasic unconventional moiety and the modified nucleotide is optionally amirror nucleotide.

In various embodiments the antisense strand (N)x comprises 2′O-Memodified ribonucleotides at the odd numbered positions (5′ to 3′;positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19). In some embodiments (N)xfurther comprises 2′O-Me modified ribonucleotides at one or bothpositions 2 and 18. In other embodiments (N)x comprises 2′O Me modifiedribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17, 19.

Other embodiments of Structures (L) are envisaged wherein x=y=21 orwherein x=y=23; in these embodiments the modifications for (N′)ydiscussed above instead of being in positions 17 and 18 are in positions19 and 20 for 21-mer oligonucleotide and 21 and 22 for 23-meroligonucleotide; similarly the modifications in positions 15, 16, 17, 18or 19 are in positions 17, 18, 19, 20 or 21 for the 21-meroligonucleotide and positions 19, 20, 21, 22, or 23 for the 23-meroligonucleotide. The 2′O Me modifications on the antisense strand aresimilarly adjusted. In some embodiments (N)x comprises 2′O Me modifiedribonucleotides at the odd numbered positions (5′ to 3′; positions 1, 3,5, 7, 9, 12, 14, 16, 18, 20 for the 21-mer oligonucleotide [nucleotideat position 11 unmodified] and 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23for the 23-mer oligonucleotide [nucleotide at position 12 unmodified].In other embodiments (N)x comprises 2′O Me modified ribonucleotides atpositions 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 [nucleotide at position 11unmodified for the 21-mer oligonucleotide and at positions 2, 4, 6, 8,10, 13, 15, 17, 19, 21, 23 for the 23-mer oligonucleotide [nucleotide atposition 12 unmodified].

In some embodiments (N′)y further comprises a 5′ terminal capnucleotide. In various embodiments the terminal cap moiety is selectedfrom an abasic unconventional moiety, an inverted abasic unconventionalmoiety, an L-DNA nucleotide, and a C6-imine phosphate (C6 amino linkerwith phosphate at terminus).

In other embodiments the present invention provides a compound havingStructure (M) set forth below:

-   (M) 5′ (N)_(x)-Z 3′ (antisense strand)    -   3′ Z′-(N′)_(y) 5′ (sense strand)        wherein each of N and N′ is selected from a pseudo-nucleotide        and a nucleotide;        wherein each nucleotide is selected from an unmodified        ribonucleotide, a modified ribonucleotide, an unmodified        deoxyribonucleotide and a modified deoxyribonucleotide;        wherein each of (N)_(x) and (N′)_(y) is an oligonucleotide in        which each consecutive N or N′ is joined to the next N or N′ by        a covalent bond;        wherein Z and Z′ are absent;        wherein x=18 to 27;        wherein y=18 to 27;        wherein the sequence of (N′)_(y) is a sequence having        complementarity to (N)x; and wherein the sequence of (N)_(x)        comprises an antisense sequence having complementarity to from        about 18 to about 27 consecutive ribonucleotides in an mRNA of a        target gene associated with acute kidney injury.

In other embodiments the present invention provides a double strandedcompound having Structure (N) set forth below:

-   -   (N) 5′ (N)_(x)-Z 3′ (antisense strand)        -   3′ Z′-(N′)_(y) 5′ (sense strand)            wherein each of N and N′ is a nucleotide selected from an            unmodified ribonucleotide, a modified ribonucleotide, an            unmodified deoxyribonucleotide and a modified            deoxyribonucleotide;            wherein each of (N)_(x) and (N′)_(y) is an oligonucleotide            in which each consecutive N or N′ is joined to the next N or            N′ by a covalent bond;            wherein Z and Z′ are absent;            wherein each of x and y is an integer between 18 and 40;            wherein the sequence of (N′)_(y) is a sequence having            complementarity to (N)x; and wherein the sequence of (N)_(x)            comprises an antisense sequence having complementarity to            from about 18 to about 40 consecutive ribonucleotides in an            antisense sequence to the mRNA of a target gene associated            with acute kidney injury;            wherein (N)x, (N′)y or (N)x and (N′)y comprise non            base-pairing modified nucleotides such that (N)x and (N′)y            form less than 15 base pairs in the double stranded            compound.

In other embodiments the present invention provides a compound havingStructure (O) set forth below:

-   (O) 5′ (N)_(x)-Z 3′ (antisense strand)    -   3′ Z′-(N′)_(y) 5′ (sense strand)        wherein each of N is a nucleotide selected from an unmodified        ribonucleotide, a modified ribonucleotide, an unmodified        deoxyribonucleotide and a modified deoxyribonucleotide;        wherein each of N′ is a nucleotide analog selected from a six        membered sugar nucleotide, seven membered sugar nucleotide,        morpholino moiety, peptide nucleic acid and combinations        thereof;        wherein each of (N)_(x) and (N′)_(y) is an oligonucleotide in        which each consecutive N or N′ is joined to the next N or N′ by        a covalent bond;        wherein Z and Z′ are absent;        wherein each of x and y is an integer between 18 and 40;        wherein the sequence of (N′)_(y) is a sequence having        complementarity to (N)x; and wherein the sequence of (N)_(x)        comprises an antisense sequence having complementarity to from        about 18 to about 40 consecutive ribonucleotides in an mRNA of a        target gene associated with acute kidney injury.

In other embodiments the present invention provides a compound havingStructure (P) set forth below:

-   -   (P) 5′ (N)_(x)-Z 3′ (antisense strand)        -   3′ Z′-(N′)_(y) 5′ (sense strand)            wherein each of N and N′ is a nucleotide selected from an            unmodified ribonucleotide, a modified ribonucleotide, an            unmodified deoxyribonucleotide and a modified            deoxyribonucleotide;            wherein each of (N)_(x) and (N′)_(y) is an oligonucleotide            in which each consecutive N or N′ is joined to the next N or            N′ by a covalent bond;            wherein Z and Z′ are absent;            wherein each of x and y is an integer between 18 and 40;            wherein one of N or N′ in an internal position of (N)x or            (N′)y or one or more of N or N′ at a terminal position of            (N)x or (N′)y comprises an abasic moiety or a 2′ modified            nucleotide;            wherein the sequence of (N′)_(y) is a sequence having            complementarity to (N)x; and wherein the sequence of (N)_(x)            comprises an antisense sequence having complementarity to            from about 18 to about 40 consecutive ribonucleotides in an            mRNA of a target gene associated with acute kidney injury.

In various embodiments (N′)y comprises a modified nucleotide at position15 wherein the modified nucleotide is selected from a mirror nucleotideand a deoxyribonucleotide.

In certain embodiments (N′)y further comprises a modified nucleotide atposition 2 wherein the modified nucleotide is selected from a mirrornucleotide and an abasic unconventional moiety.

In various embodiments the antisense strand (N)x comprises 2′O-Memodified ribonucleotides at the odd numbered positions (5′ to 3′;positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19). In some embodiments (N)xfurther comprises 2′O-Me modified ribonucleotides at one or bothpositions 2 and 18. In other embodiments (N)x comprises 2′O Me modifiedribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17, 19.

An additional novel molecule provided by the present invention is anoligonucleotide comprising consecutive nucleotides wherein a firstsegment of such nucleotides encode a first inhibitory RNA molecule, asecond segment of such nucleotides encode a second inhibitory RNAmolecule, and a third segment of such nucleotides encode a thirdinhibitory RNA molecule. Each of the first, the second and the thirdsegment may comprise one strand of a double stranded RNA and the first,second and third segments may be joined together by a linker. Further,the oligonucleotide may comprise three double stranded segments joinedtogether by one or more linker.

Thus, one molecule employed in the methods of the present invention isan oligonucleotide comprising consecutive nucleotides which encode threeinhibitory RNA molecules; said oligonucleotide may possess a triplestranded structure, such that three double stranded arms are linkedtogether by one or more linker, such as any of the linkers presentedhereinabove. This molecule forms a “star”-like structure, and may alsobe referred to herein as RNAstar and described in PCT Patent PublicationWO 2007/091269 assigned to one of the assignees of the presentapplication.

Said triple-stranded oligonucleotide may be an oligoribonucleotidehaving the general structure:

5′ Oligo1 (sense) LINKER A Oligo2 (sense) 3′ 3′ Oligo1 (antisense)LINKER B Oligo3 (sense) 5′ 3′ Oligo3 (antisense) LINKER C Oligo2(antisense) 5′ or 5′ Oligo1 (sense) LINKER A Oligo2 (antisense) 3′ 3′Oligo1 (antisense) LINKER B Oligo3 (sense) 5′ 3′ Oligo3 (antisense)LINKER C Oligo2 (sense) 5′ or 5′ Oligo1 (sense) LINKER A Oligo3(antisense) 3′ 3′ Oligo1 (antisense) LINKER B Oligo2 (sense) 5′ 5′Oligo3 (sense) LINKER C Oligo2 (antisense) 3′wherein one or more of linker A, linker B or linker C is present; anycombination of two or more oligonucleotides and one or more of linkersA-C is possible, so long as the polarity of the strands and the generalstructure of the molecule remains. Further, if two or more of linkersA-C are present, they may be identical or different.

Thus, a triple-armed structure is formed, wherein each arm comprises asense strand and complementary antisense strand (i.e. Oligo 1 antisensebase pairs to Oligo 1 sense etc.). The triple armed structure may betriple stranded, whereby each arm possesses base pairing.

Further, the above triple stranded structure may have a gap instead of alinker in one or more of the strands. Such a molecule with one gap istechnically quadruple stranded and not triple stranded; insertingadditional gaps or nicks will lead to the molecule having additionalstrands. Preliminary results obtained by the inventors of the presentinvention indicate that said gapped molecules are more active ininhibiting certain target genes than the similar but non-gappedmolecules. This may also be the case for nicked molecules.

According to one preferred embodiment of the invention, the antisenseand the sense strands of the siRNA are phosphorylated only at the3′-terminus and not at the 5′-terminus. According to another preferredembodiment of the invention, the antisense and the sense strands arenon-phosphorylated. According to yet another preferred embodiment of theinvention, the 5′ most ribonucleotide in the sense strand is modified toabolish any possibility of in vivo 5′-phosphorylation.

The invention further provides a vector capable of expressing any of theaforementioned oligoribonucleotides in a cell after which appropriatemodification may be made. In preferred embodiment the cell is amammalian cell, preferably a human cell.

Methods of Treatment

In one embodiment, the present invention relates to a method for thetreatment of a subject in need of treatment for attenuation of CKDprogression which is associated with expression of one or more of thetarget genes of Table 1, comprising administering to the subject anamount of an oligonucleotide inhibitor, which reduces, down regulates orinhibits expression or upregulation of one or more of those genes.

A number of conditions can cause permanent damage to the kidneys and/oraffect the function of the kidneys and lead to CKD. The most commoncauses of CKD in adults are:

-   -   a) Diabetes. Diabetic nephropathy (DN) is a common complication        of diabetes;    -   b) High blood pressure. Untreated or poorly treated high blood        pressure is a major cause of CKD;    -   c) Aging kidneys. There appears to be an age-related decline in        kidney function;    -   d) Acute or chronic kidney ischemia (this is the model we used        in rats and you are using in humans);    -   e) sepsis.

Other less common conditions that can lead to CKD include diseases ofthe glomeruli, such as glomerulonephritis (inflammation of the glomeruliin the kidneys); renal artery stenosis (narrowing), haemolytic-uraemicsyndrome, polycystic kidney disease, blockages to the flow of urine,drug and toxin-induced kidney damage, and repeated kidney infections.

In some embodiments, the present invention relates to a method for thetreatment of a subject in need of treating chronic kidney disease (CKD)which is associated with expression of one or more of the target genesof Table 1, supra, comprising administering to the subject an amount ofan oligonucleotide inhibitor, which prevents upregulation oroverexpression of one or more of those genes in a kidney of the subject.In certain embodiments the upregulation or overexpression of one or moreof the target genes is in response to renal insult or injury. In someembodiments the renal insult is an acute renal insult including acutekidney injury (AKI). In various embodiments of the invention treatmentincludes preventing or delaying onset of CKD and preventing exacerbationand progression of CKD.

In preferred embodiments the subject being treated is a warm-bloodedanimal and, in particular, mammals including non-human primate andhuman.

The methods of the invention comprise administering to the subject oneor more inhibitory compounds which down-regulate expression of thetarget genes of Table 1; and in particular siRNA in a therapeuticallyeffective dose so as to thereby treat the subject.

In various embodiments the inhibitor is selected from the groupconsisting of siRNA, shRNA, an aptamer, an antisense molecule, miRNA,and a ribozyme. In the presently preferred embodiments the inhibitor issiRNA. In preferred embodiments the siRNA is a chemically synthesized,modified compound.

The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor delay the onset of CKD, attenuate, prevent or slow down CKD orprogression or severity of CKD as listed above. Those in need oftreatment include those already experiencing the disease or condition,those prone to CKD, and those in which CKD is to be prevented; forexample in a subject exposed to repetitive renal insults, includingrenal insults due to nephrotoxic drugs, such as, without being limitedto, antibiotics (e.g. aminoglycosides), chemotherapeutic drugs (e.g.Cisplatin), immunosuppressant drugs (e.g. Cyclosporin A, Tacrolimus(also FK-506 or Fujimycin)) and radiocontrast agents, orischemia-reperfusion injury (IRI). According to various embodiments ofthe present invention the oligonucleotide inhibitor is administeredbefore, during or subsequent to the exposure to the renal insult,preferably subsequent to the insult. In some embodiments theoligonucleotide inhibitor is a siRNA compound. In various embodimentsthe siRNA is administered to the subject at about 4 hours post renalinsult. In cases where treatment is for the purpose of prevention, thenthe present invention relates to a method for delaying the onset of oraverting the development of the CKD. In some embodiments CKD develops inresponse to repetitive renal insults including repetitive acute kidneyinjury (AKI).

Acute renal failure (ARF), also known as acute kidney injury (AKI), is arapid loss of renal function due to kidney damage and resulting inretention of nitrogenous (urea and creatinine) and non-nitrogenous wasteproducts in the blood. Depending on the severity and duration of therenal dysfunction, this accumulation is accompanied by metabolicdisturbances, such as metabolic acidosis (acidification of the blood)and hyperkalaemia (elevated serum potassium levels), changes in bodyfluid balance, and effects on many other organ systems. It can becharacterized by oliguria or anuria (decrease or cessation of urineproduction).

Glomerular filtration rate “GFR” describes the flow rate of filteredfluid across the glomeruli. The assessment of GFR is the most commonlyused test of renal function. In some embodiments the method ofattenuating progression of CKD or preventing exacerbation of CKD ismeasured as an increase of about 5%, 10%, 20%, 30%, 40% or more in GFRin a treated subject when compared to the untreated subject.

Creatinine clearance rate (CCr, mL/min/1.73 m²) is the volume of bloodplasma that is cleared of creatinine per unit time and is a usefulmeasure for approximating the true GFR.

SCr (mg/dL) relates to serum creatinine levels. In some embodiments themethod of attenuating progression of CKD or preventing exacerbation ofCKD is measured as an decrease of about 5%, 10%, 20%, 30%, 40% or morein SCr in a treated subject when compared to the untreated subject.

In some embodiments the method of the invention relates to a method oftreating CKD induced by repetitive acute kidney injury (AKI) insults, inparticular acute renal failure due to ischemia in post surgicalpatients, acute renal failure due to chemotherapy treatment such ascisplatin administration, sepsis-associated acute renal failure,nephrotoxin induced AKI including radiocontrast media induced AKI.Contrast induced AKI (CIAKI) (also known as contrast-inducednephropathy) relates to the induction of AKI by intravascularadministration of iodinated contrast media, for example in patientsundergoing angiography, and in particular coronary angiography. Inanother embodiment the method of the invention relates to the preventionof CKD in high-risk patients undergoing major cardiac surgery orvascular surgery. The patients are at risk of developing acute renalfailure which in some cases progresses to CKD. Those patients areidentified using various scoring methods such as the Cleveland Clinicalgorithm or that developed by US Academic Hospitals (QMMI) and byVeterans' Administration (CICSS).

In another preferred embodiment, the methods of the present inventionrelate to treating or preventing CKD in a subject induced by treatmentof the subject with a nephrotoxin including a diuretic, a β-blocker, avasodilator agent, an ACE inhibitor, an immunosuppressant (e.g.cyclosporin), an aminoglycoside antibiotic (e.g. gentamicin), anantifungal (e.g. amphotericin B), a chemotherapeutic agent (e.g.cisplatin), radiocontrast media, an antibody (e.g. immunoglobulin),mannitol, a NSAID (e.g. aspirin, ibuprofen, diclofenac),cyclophosphamide, methotrexate, aciclovir, polyethylene glycol, β-lactamantibiotics, vancomycin, rifampicin, sulphonamides, ciprofloxacin,ranitidine, cimetidine, furosemide, thiazides, phenytoin, penicillamine,lithium salts, fluoride, demeclocycline, foscarnet, aristolochic acid,an anti-oxidant, a calcium channel blocker, a vasoactive substance, agrowth factors, an anti-inflammatory agents and more.

In the majority of hospitalized ARF patients, ARF is caused by acutetubular necrosis (ATN), which results from ischemic, septic and/ornephrotoxic insults. Renal hypoperfusion is caused by hypovolemic,cardiogenic and septic shock, by administration of vasoconstrictivedrugs or renovascular injury. Nephrotoxins include exogenous toxins,such as radiocontrast media, aminoglycosides and cisplatin andcisplatin-like compounds, as well as endogenous toxins, such asmyoglobin. Without wishing to be bound to theory, recent studies supportthe theory that apoptosis in renal tissues is prominent in most humancases of ARF. The principal site of apoptotic cell death is the distalnephron. During the initial phase of ischemic injury, loss of integrityof the actin cytoskeleton leads to flattening of the epithelium, withloss of the brush border, loss of focal cell contacts, and subsequentdisengagement of the cell from the underlying substratum. It has beensuggested that apoptotic tubule cell death may be more predictive offunctional changes than necrotic cell death (Komarov et al., Science1999, 10; 285(5434):1733-7); Supavekin et al., Kidney Int. 2003,63(5):1714-24).

A “contrast agent,” as used herein, refers to a compound employed toimprove the visibility of internal body structures in an image, such asan X-ray image or a scanning image (e.g., CAT (Computerized AxialTomography) scan, MRI (Magnetic Resonance Imaging) scan). The termcontrast agent is also referred to herein as a radiocontrast agent.Contrast agents are employed in various diagnostic (e.g. embolism;cardiac catheterization) and therapeutic procedures. Contrast-inducednephropathy (CIN) remains the primary risk factor in the use of contrastagents. Patients with pre-existing renal failure and diabetes are atparticularly high risk. Moreover, CIN is associated with significantin-hospital and long-term morbidity and mortality.

Additional mechanisms that contribute to the development of AKI:ischemia, vasoconstriction, toxic injury related to selected endogenoussubstances (e.g. myoglobin in rhabdomyolysis due to crush injury andextensive blunt trauma), radiocontrast (iodinated and IV contrast forradiological examination including CT angiography, cardiacarteriography), phosphate nephropathy due to bowel preparation forcolonoscopy with sodium phosphate, nephrotoxic drugs (e.g., NSAIDs,aminoglycoside antibiotics, gentamycin and penicillin, amphotericin B),microcirculatory changes, as observed with sepsis and other inflammatorystates, hemolysis, diagnostic cardiac catheterization, femoralarteriography especially in aged or diabetic patients, percutaneouscoronary intervention (PCI), coronary artery bypass grafting (CABG),sepsis, thoracoabdominal aortic surgery, aortic aneurysim repair forexample for infra-renal aortic abdominal surgery or thoracic orthoracoabdominal aortic surgery.

Preexisting conditions predicting severity and long term outcome of AKIpatients with coronary arthery disease (CAD), heart failure, diabetes,vascular complications (e.g. atheroembolic disease and renal veinthrombosis), HIV-infected patients, gender, older age (>60),pre-existing chronic kidney disease or underlying renal insufficiency,volume depletion, hepatitis co-infection, liver disease, hepatorenalsyndrome, cancer patients, patients with serious water and electrolytemetabolism disturbances, patients with hematological andnon-hematological malignancies, cirrhosis, COPD, severe burns,pericarditis and pancreatitis.

Intrinsic damage to the kidney:

toxins or medication (e.g. some NSAIDs, aminoglycoside antibiotics,iodinated contrast, lithium, phosphate nephropathy due to bowelpreparation for colonoscopy with sodium phosphates)rhabdomyolysis (breakdown of muscle tissue)—the resultant release ofmyoglobin in the blood affects the kidney; it can be caused by injury(especially crush injury and extensive blunt trauma), statins,stimulants and some other drugshemolysis (breakdown of red blood cells)—the hemoglobin damages thetubules; it may be caused by various conditions such as sickle-celldisease, and lupus erythematosusmultiple myeloma, either due to hypercalcemia or “cast nephropathy”(multiple myeloma can also cause chronic renal failure by a differentmechanism)acute glomerulonephritis which may be due to a variety of causes, suchas anti glomerular basement membrane disease/Goodpasture's syndrome,Wegener's granulomatosis or acute lupus nephritis with systemic lupuserythematosusPost-renal (obstructive causes in the urinary tract) due to medicationinterfering with normal emptying of bladder (e.g. anticholinergics),benign prostatic hypertrophy or prostate cancer, kidney stones,abdominal malignancy (e.g. ovarian cancer, colorectal cancer),obstructed urinary catheter, drugs that can cause crystalluria and drugsthat can lead to myoglobinuria & cystitis.

In conclusion, currently there is no satisfactory mode of therapy forthe prevention and/or treatment of CKD induced by recurring acuteinsults to the kidney, and there is a need therefore to develop novelcompounds for this purpose.

Pharmaceutical Compositions

While it may be possible for the compounds of the present invention tobe administered as the raw chemical, it is preferable to present them asa pharmaceutical composition. Accordingly the present invention providesa method employing a pharmaceutical composition comprising one or moreof the oligonucleotide compounds of the invention; and apharmaceutically acceptable carrier. This composition may comprise amixture of two or more different siRNA compounds.

In some embodiments the pharmaceutical composition comprises at leastone siRNA compound of the invention covalently or non-covalently boundto one or more siRNA compounds of the invention in an amount effectiveto inhibit the target genes of the present invention; and apharmaceutically acceptable carrier. The compound may be processedintracellularly by endogenous cellular complexes to produce one or moreoligoribonucleotides of the invention.

The invention further provides a pharmaceutical composition comprising apharmaceutically acceptable carrier and one or more of the compounds ofthe invention in an amount effective to inhibit expression in a cell ofa human target gene of the present invention, the compound comprising asequence (N). which is substantially complementary to the sequence of atarget nucleic acid.

“Having complementarity” or “substantially complementary” refers tocomplementarity of greater than about 84%, to another sequence. Forexample in a duplex region consisting of 19 base pairs one mismatchresults in 94.7% complementarity, two mismatches results in about 89.5%complementarity and 3 mismatches results in about 84.2% complementarity,rendering the duplex region substantially complementary. Accordinglysubstantially identical refers to identity of greater than about 84%, toanother sequence.

Additionally, the invention provides a method of inhibiting theexpression of the target genes of the present invention by at least 40%,preferably by 50%, 60% or 70%, more preferably by 75%, 80% or 90% ascompared to a control comprising contacting an mRNA transcript of thetarget gene of the present invention with one or more of the compoundsof the invention.

In one embodiment the oligoribonucleotide is inhibiting one or more ofthe target genes of the present invention, whereby the inhibition isselected from the group comprising inhibition of gene function,inhibition of polypeptide and inhibition of mRNA expression.

In one embodiment the compound inhibits the target polypeptide, wherebythe inhibition is selected from the group comprising inhibition offunction (which may be examined by an enzymatic assay or a binding assaywith a known interactor of the native gene/polypeptide, inter alia),inhibition of protein (which may be examined by Western blotting, ELISAor immuno-precipitation, inter alia) and inhibition of mRNA expression(which may be examined by Northern blotting, quantitative RT-PCR,in-situ hybridisation or microarray hybridisation, inter alia).

Additionally, the invention provides a method of treating or preventingkidney damage in a subject at risk of CKD associated with activation orupregulation or overexpression of one or more of the target genes of thepresent invention, the method comprising administering to the subject acompound of the invention in a therapeutically effective dose therebytreating or preventing kidney damage in the subject.

In additional embodiments the invention provides a method of treating asubject at risk of developing CKD accompanied by or associated with orresulting from an elevated level of one or more of the target genes ofthe present invention, the method comprising administering to thesubject a compound of the invention in a therapeutically effective dosethereby reducing the risk of developing CKD in the subject.

Delivery

The siRNA compound useful in methods of the invention is administered asthe compound per se (i.e. as naked siRNA) or as pharmaceuticallyacceptable salt and is administered alone or as an active ingredient incombination with one or more pharmaceutically acceptable carrier,solvent, diluent, excipient, adjuvant and vehicle. In some embodiments,the siRNA molecules useful in methods of the present invention aredelivered to the target tissue by direct application of the nakedmolecules prepared with a carrier or a diluent.

The term “naked siRNA” refers to siRNA molecules that are free from anydelivery vehicle that acts to assist, promote or facilitate entry intothe cell, including viral sequences, viral particles, liposomeformulations, lipofectin or precipitating agents and the like. Forexample, siRNA in PBS is “naked siRNA”.

However, in some embodiments the siRNA molecules of the invention aredelivered in liposome formulations and lipofectin formulations and thelike and can be prepared by methods well known to those skilled in theart. Such methods are described, for example, in U.S. Pat. Nos.5,593,972, 5,589,466, and 5,580,859, which are herein incorporated byreference.

Delivery systems aimed specifically at the enhanced and improveddelivery of siRNA into mammalian cells have been developed, (see, forexample, Shen et al., FEBS Let. 2003, 539:111-115; Xia et al., Nat.Biotech. 2002, 20:1006-1010; Reich et al., Mol. Vision 2003, 9: 210-216;Sorensen et al., J. Mol. Biol. 2003. 327: 761-766; Lewis et al., Nat.Gen. 2002, 32: 107-108 and Simeoni et al., NAR 2003, 31, 11: 2717-2724).siRNA has recently been successfully used for inhibition of geneexpression in primates (see for example, Tolentino et al., Retina24(4):660).

The pharmaceutically acceptable carriers, solvents, diluents,excipients, adjuvants and vehicles as well as implant carriers generallyrefer to inert, non-toxic solid or liquid fillers, diluents orencapsulating material not reacting with the active ingredients of theinvention and they include liposomes and microspheres. Examples ofdelivery systems useful in the present invention include U.S. Pat. Nos.5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603;4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196. Other suchimplants, delivery systems, and modules are well known to those skilledin the art. The siRNAs or pharmaceutical compositions of the presentinvention are administered and dosed in accordance with good medicalpractice, taking into account the clinical condition of the individualsubject, the disease to be treated, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners.

The “therapeutically effective dose” for purposes herein is thusdetermined by such considerations as are known in the art. The dose mustbe effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

In general, the active dose of compound for humans is in the range offrom 1 ng/kg to about 20-100 mg/kg body weight per day, preferably about0.01 mg to about 2-10 mg/kg body weight per day, in a regimen of asingle dose or multiple doses, e.g. or two doses or three or more doses,administered within 24 hours of each renal insult. The siRNA compoundsuseful in methods of the present invention can be administered by any ofthe conventional routes of administration. The compounds areadministered orally, subcutaneously or parenterally includingintravenous, intraarterial, intramuscular, intraperitoneally, andintranasal administration as well as intrathecal and infusiontechniques. Implants of the compounds are also useful. Liquid forms areprepared for injection, the term including subcutaneous, transdermal,intravenous, intramuscular, intrathecal, and other parental routes ofadministration. The liquid compositions include aqueous solutions, withand without organic co-solvents, aqueous or oil suspensions, emulsionswith edible oils, as well as similar pharmaceutical vehicles. In aparticular embodiment, the administration comprises intravenousadministration.

Pharmaceutical compositions that include the nucleic acid moleculedisclosed herein may be administered once daily, qid, tid, bid, QD, orat any interval and for any duration that is medically appropriate.However, the composition may also be dosed in dosage units containingtwo, three, four, five, six or more sub-doses administered atappropriate intervals throughout the day.

In some embodiments the dosage unit is compounded for a single dose overseveral days, e.g., using a conventional sustained release formulationwhich provides sustained and consistent release of the dsRNA over aseveral day period. Sustained release formulations are well known in theart. In certain embodiments the methods of the invention includeadministering one or more siRNA compound or compounds to the subject forsustained or controlled delivery. The methods of the present inventionrely primarily on parenteral administration routes and more specificallyon implant depots or depot injections, which provide for prolongedrelease of the biological agent into the circulatory system. Devices foruse in these parenteral delivery systems include non-injectable andinjectable devices. Non-injectable devices include an implant such as asiRNA depot implant, or similar device. Known depot implants include,but are not limited to, synthetic and natural materials including solidbiodegradable and non-biodegradable polymer devices including foams,gels, matrices, and the like comprising one or more of dextran, fibrin,hyaluronate, chitosan and the like as well as a pump and micropumpsystems also known in the art. Injectable devices include bolusinjections (release and dissipation of the compound subsequent toinjection), and repository or depot injections, which provide a storagereservoir or depot at the site of injection, allowing for sustainedrelease of the biological agent over time.

The present invention also provides for a process of preparing apharmaceutical composition useful in a method according to presentinvention, which comprises:

-   -   providing one or more double stranded compound of the invention;        and    -   admixing said compound with a pharmaceutically acceptable        carrier.

The present invention also provides for a process of preparing apharmaceutical composition, useful in a method according to presentinvention, which comprises admixing one or more siRNA compoundsaccording to present invention with a pharmaceutically acceptablecarrier.

In a preferred embodiment, the siRNA compound used in the preparation ofa pharmaceutical composition, useful in a method according to presentinvention, is admixed with a carrier in a pharmaceutically effectivedose. In a particular embodiment the siRNA compound, useful in a methodof the present invention is conjugated to a steroid, vitamin or to alipid or to another suitable molecule e.g. to cholesterol.

The present invention is illustrated in detail below with reference toexamples, but is not to be construed as being limited thereto.

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

EXAMPLES General Methods in Molecular Biology

Standard molecular biology techniques known in the art and notspecifically described were generally followed as in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York (1989), and as in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and as inPerbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, NewYork (1988), and as in Watson et al., Recombinant DNA, ScientificAmerican Books, New York and in Birren et al (eds) Genome Analysis: ALaboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press,New York (1998) and methodology as set forth in U.S. Pat. Nos.4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 andincorporated herein by reference. Polymerase chain reaction (PCR) wascarried out generally as in PCR Protocols: A Guide To Methods AndApplications, Academic Press, San Diego, Calif. (1990). In situ (Incell) PCR in combination with Flow Cytometry can be used for detectionof cells containing specific DNA and mRNA sequences (Testoni et al.,1996, Blood 87:3822.) Methods of performing RT-PCR are also well knownin the art.

Example 1 Generation of Sequences for Active siRNA Compounds to TargetGenes and Production of the siRNAs

Using proprietary algorithms and the known sequence of the target genes,the sequences of many potential siRNAs were generated. In addition tothe algorithm, some of the 23-mer oligomer sequences were generated by5′ and/or 3′ extension of the 19-mer sequences. The sequences that havebeen generated using this method are fully complementary to thecorresponding mRNA sequence.

Sequence listing: The sequence listing for this application (SEQ IDNO:1-SEQ ID NO:119) has been submitted electronically as sequencelisting file entitled “209-PCT1_ST25.txt” created Jun. 7, 2010, 459 kb.Applicants hereby incorporate by reference the sequence listing into theinstant specification.

Example 2 In Vitro Testing of siRNA Compounds 1. General

About 1.5-2×10⁵ test cells (HEPG2 or PC3 cells for siRNA targeting thehuman gene) were seeded per well in 6 wells plate (70-80% confluent).

After 24 h cells were transfected with siRNA oligomers usingLipofectamine™ 2000 reagent (Invitrogene) at final concentration of 500pM, 5 nM, 20 nM or 40 nM. The cells were incubated at 37° C. in a CO₂incubator for 72 h.

As positive control for cells transfection PTEN-Cy3 labeled siRNA oligoswere used. As negative control for siRNA activity GFP siRNA oligos wereused.

About 72 h after transfection cells were harvested and RNA was extractedfrom cells. Transfection efficiency was tested by fluorescentmicroscopy.

The percent of inhibition of gene expression using specific siRNAs wasdetermined using qPCR analysis of target gene in cells expressing theendogenous gene.

The inhibitory activity of the compounds of the present invention ontarget genes or binding of the compounds of the present invention totarget genes may be used to determine the interaction of an additionalcompound with the target polypeptide, e.g., if the additional compoundcompetes with the oligonucleotides of the present invention forinhibition of a target gene, or if the additional compound rescues saidinhibition. The inhibition or activation is tested by various means,such as, inter alia, assaying for the product of the activity of thetarget polypeptide or displacement of binding compound from the targetpolypeptide in radioactive or fluorescent competition assays.

Example 3 siP53 Compounds

QM5 is a chemically modified siRNA compound which targets rat and mousep53 and is the disclosed of International Patent Publication WO2006/035434, assigned to one of the assignees of the present invention.QM5. The compound has two separate strands, sense (SEN; passenger) andantisense (AS; guide), each comprising alternating unmodifiedribonucleotides (upper case letters) and 2′-methoxy (2′-O-Me; 2′-O—CH₃)sugar modified ribonucleotides (lower case letters) on both strandsforming a specific pattern as shown herein below:

Sense (passenger) sequence 5′ GaAgAaAaUuUcCgCaAaA 3′ (SEQ ID NO: 116Antisense (guide) sequence 3′ cUuCuUuUaAaGgCgUuUu 5′ (SEQ ID NO: 117)

The I5 compound is a 19-mer blunt-ended nucleic acid duplex that targetshuman p53, a gene that plays a pivotal role in the stress-responseapoptotic pathway. The compound has two separate strands, sense (SEN)and antisense (AS), each comprising alternating unmodifiedribonucleotides (upper case letters) and 2′-methoxy (2′OMe) sugarmodified ribonucleotides (lower case letters) on both strands forming aspecific pattern as shown herein below:

Sense (passenger) sequence 5′ GaGaAuAuUuCaCcCuUcA 3′ (SEQ ID NO: 118)Antisense (guide) sequence 3′ cUcUuAuAaAgUgGgAaGu 5′ (SEQ ID NO: 119)

For treating or preventing kidney damage in a human subject at risk ofchronic kidney disease (CKD) associated with exposure to a recurrence ofrenal insults a therapeutically effective dose of I5 is administered tothe subject within 24 hours of each renal insult thereby treating CKD.15 is the subject of WO 2006/035434, assigned to one of the assignees ofthe present invention.

Example 4 Model Systems of CKD

The following animal models were implemented to support the methods ofthe invention

(1) preventing CKD development in a subject at risk of CKD due tomultiple renal insults (supported by example 4-1;(2) preventing acceleration/progression of CKD development by AKIepisodes in a subject with a CKD background (supported by Example 4-2);and(3) attenuating the severity of AKI on the background of CKD (supportedby Example 4-2).

Example 4-1 Bilateral Kidney Arterial Clamp CKD Model

This animal model is useful in assessing the test compounds forprevention of CKD or attenuation of CKD progression resulting fromrepetitive AKI/ARF insults.

Repetitive AKI/ARF insults often results in the exacerbation of chronickidney disease (CKD), progression of CKD or development of CKD (see FIG.1). ARF is a clinical syndrome characterized by rapid deterioration ofrenal function that occurs within days. Without being bound by theorythe acute kidney injury may be the result of renal ischemia-reperfusioninjury such as renal ischemia-reperfusion injury in patients undergoingmajor surgery such as major cardiac surgery. The principal feature ofARF is an abrupt decline in glomerular filtration rate (GFR), resultingin the retention of nitrogenous wastes (urea, creatinine) in the blood.Recent studies, support that apoptosis in renal tissues is prominent inmost human cases of ARF. The principal site of apoptotic cell death isthe distal nephron. During the initial phase of ischemic injury, loss ofintegrity of the actin cytoskeleton leads to flattening of theepithelium, with loss of the brush border, loss of focal cell contacts,and subsequent disengagement of the cell from the underlying substratum.

The rat model for CKD comprises repetitive (5 times)ischemia-reperfusion-induced ARF as follows and as shown in FIG. 2:

Ischemia-reperfusion injury was induced in rats following 45 minutesbilateral kidney arterial clamp and subsequent release of the clamp toallow 24 hours of reperfusion. PBS or QM5 (rat siP53) (12 mg/kg) wereinjected i.v. into individual experimental animals 4 hours post clamp.ARF progression was monitored by measurement of serum creatinine (SCr)levels before (baseline) and 24 hrs, 2 days and 7 days post surgery. Thetreatment (I/R injury, QM5, SCr measurement) was repeated for four morecycles at 30-day intervals, for a total of five cycles. At 7 days post5^(th) cycle 24 hour creatinine clearance (CrCl) metabolic cage andurine protein were measured. The right kidneys were surgically removed 2days after metabolic cage (day 10 post 5^(h) cycle) and the kidney washistologically analyzed for CKD. At 3 weeks post right nephrectomy theleft kidney was exteriorized and studied in vivo using intravitaltwo-photon microscopy (for Cy3-siRNa uptake and retention).

Results

Age-matched untreated rats had much more uniform uptake and distributionof the Cy3 labeled siRNA. Twenty-four hours after the initial injection,reduction in cellular levels of the siRNA was visible. Also tubularlumens were generally more open with only a few collapsed lumensvisible.

Saline treated CKD rats had much more heterogeneous distribution(patchy) and uptake of the siRNA. Tubules could be seen with thinepithelia and the lumen greatly distended. Uptake in tubular cells didoccur, but at a lower level than surrounding tubular cells with morenormal morphology. Degradation of the labeled siRNA appeared slower inthese rats as there appeared to be greater residual fluorescence than inage-matched untreated rats at 24 hours.

QM5 treated CKD rats displayed uptake and distribution characteristicsthat were intermediary between the control age-matched untreated andsaline treated CKD groups. Overall uptake was more homogeneous whenviewing individual fields. Cystic tubules were still present onoccasion. Overall, QM5 aided in the uniform delivery of the Cy3-labeledsiRNA to the tubular epithelia, this was more readily apparent in Rat#4. Metabolism of siRNA at 24 hr was also intermediate betweenage-matched untreated rats and saline treated ischemic rats. Underphysiological conditions, in age-matched untreated rats Cy3-labeledsiRNA, following intravenous injection, was rapidly filtered across theglomerulus and taken up selectively by proximal tubule cells (PTC).Total cellular and cytosolic accumulation in proximal tubule cells wasquantified using threshold analysis and revealed a maximum at 120minutes with a rapid decay over the next four hours. The biologicalactivity of the siRNA correlated closely to the fluorescent half life.

FIG. 3 shows the effect of p53 siRNA on kidney function followingrepetitive ischemic injury. Serum creatinine levels prior to eachischemia (AKI) cycle, and at days 1, 2, and 7 post each ischemia cyclein rats treated with PBS or siP53 (QM5) (12 mg/kg) given i.v. at 4 hrspost each ischemia (AKI). Data represent the mean+SD (n=10/group).

FIG. 4 shows that siP53 protects GFR and minimizes proteinuria.Measurements from PBS treated animals are shown by hatched columns,while QM5 treated animals are solid columns.

Table 2 hereinbelow shows the effect of siP53 on kidney functionfollowing five monthly cycles of ischemic injury: siP53 protectsglomerular filtration rate and minimizes proteinuria. Glomerularfiltration rate (GFR) and proteinuria (Uprot) were measured at 7 daysfollowing last (5th) AKI cycle by 24 hrs urine collection and tail bloodcollection. Groups: PBS—rats were treated with PBS given i.v. at 4 hrspost each ischemic injury; QM5—siP53 (12 mg/kg) given i.v. at 4 hrs posteach ischemic injury. Data represent means+SD (n=10/group)

TABLE 2 Injury score, mean/group (n = 10) ± SD Histopatholgy scoringparameters PBS QM5 p-value Glomerular Damage 0.1 + 0.3 0.1 + 0.3 1Interstitial Cellular Infiltrate 1.2 + 0.4 0.5 + 0.5 0.008 Interstitialfibrosis 1.3 + 0.5 0.9 + 0.6 0.12 Tubular status 1.4 + 0.5 0.8 + 0.60.04 Vasculopathy 0.1 + 0.3 0 0.37 Total Chronic injury score 4.1 + 0.72.3 + 1.2 0.02 Tubular necrosis 0.1 + 0.3 0 0.37 Tubular dilation 1.4 +0.5 0.8 + 0.6 0.04 Casts 1.3 + 1.3 0.5 + 0.5 0.16 Total Acute injuryscore 2.8 + 1.8 1.3 + 0.9 0.05 Total pathology score 6.9 + 3.3 3.6 + 1.80.02 Chronic + acute

FIG. 5 shows histopathology scoring of right kidney procured 10 dayspost fifth AKI cycle. At 10 days post last (5th) ischemic injury, therats (treated with PBS or siP53 at 4 hrs post each monthly ischemicinjury) were subjected to right nephrectomy. Harvested right kidneysections were blindly analyzed by board-certified pathologist. At leasttwo representative kidney sections were analyzed for each rat. Theparameters of acute (tubular necrosis, tubular dilation, casts) andchronic (glomerular damage, interstitial cellular infiltrate,interstitial fibrosis, tubular status and vasculopathy) damage wereanalyzed. Total acute and total chronic injury scores are a sums of allchronic or acute damage parameters respectively. Total pathology scoreis a sum of acute and chronic injury scores for each rat. Grading ofpathological changes was performed according to the following scoringsystem:

Grade 0—no pathological changes; Grade 1—feature involves 1 to 10% ofthe area (mild and focal); Grade 2—feature involves 10 to 25% of thearea (moderate and multifocal); Grade 3—feature involves 25 to 75% ofthe area (diffuse damage without damage of normal architecture); Grade4—feature involves more than 75% of the area (diffuse damage withprominent damage of normal kidney architecture). Data represent themean+SD (n=10/group).

Example 4-2 Uninephrectomy and High Salt Diet

This animal model is useful in assessing the test compounds forreduction/attenuation of AKI/ARF in a CKD background thereby providing amodel for preventing exacerbation or progression of CKD by recurringAKI/ARF insults in CKD patients and in attenuating the severity of AKIin patients suffering from CKD who undergo a procedure or event likelyto cause AKI.

FIG. 6 shows the study design used to establish CKD. In summary, SD ratswere subjected to right nephrectomy, followed by 3 or 4 repetitivebimonthly cycles of AKI (until SCr and GFR rates were at CKD levels).The first AKI cycle comprised a left pedicle clamp for a period of 45minutes, whereas all following AKIs comprised a 30 minute clamp.Throughout the entire period, the rats were fed a high salt diet. After3 or 4 cycles, the following kidney function parameters were evaluated:serum creatinine (SCr), GFR and urine protein.

Animal was considered to have moderate to severe CKD when serumcreatinine (SCr) levels were above 0.8 mg/ml and glomerular filtrationrate (GFR) was less than 0.60 ml/min/100 gr. In addition, 3 rats wereuninephrectomized and fed with regular diet for the same total period oftime as above (7-8 months). At the end of this period, their SCr, GFRand Uprot were evaluated. Results are shown in Table 3, hereinbelow:

TABLE 3 GFR, SCr, ml/min/ Uprot, mg/ml 100 g BW mg/24 h Normal rats(historical and 0.2-0.3 0.8-0.9* published data) Uninephr rats after 3AKI cycles 1.13 ± 0.05 0.16 ± 0.03 221 ± 21  (N = 3), HS diet Uninephrrats after 4 AKI cycles 0.98 ± 0.17 0.19 ± 0.03 646 ± 160 (N = 4), HSdiet *Zaladek-Gil et al (1999), Braz J Med Biol Res, 32: 107-113;Chamberlain et al, (2007) Exp Physiol, 92: 251-262

FIG. 7 shows kidney function parameters in group sorted animals prior toAKI/ARF insults and prior to siP53 treatment). The results show thatuninephrectomized animals exposed to a high salt diet exhibit moresevere CKD than uninephrectomized animals exposed to a normal diet asmeasured by SCr, GFR and urine protein (Uprot) levels. CKD and controlanimals received siP53 or siGFP (12 mg/kg) or vehicle by i.v. injectionat 4 hours post last AKI insult. FIG. 8 shows the effect of siP53 (QM5)on prevention of AKI insult in animals with CKD.

FIGS. 9A-9H show histopathological parameters for acute injury (9A-9C)and chronic injury (9D-9H). The scoring system for histopathology was asfollows: 0: none; 1: mild and focal; 2: moderate and multifocal; 3:diffuse without damage of normal architecture; 4: diffuse with prominentdamage of normal architecture. All acute parameters, tubular necrosis,tubular dilation and urinary casts were improved in siP53 treatedanimals. The chromic injury parameters glomerular damage, interstitialinfitrate, interstitial fibrosis and tubular atrophy were reduced intreated animals.

Average histopathology scores for acute and chronic injury in treatedand untreated animals are shown in FIG. 10.

While certain embodiments of the invention have been illustrated anddescribed, it will be clear that the invention is not limited to theembodiments described herein. Modifications, changes, variations,substitutions and equivalents will be apparent to those skilled in theart without departing from scope of the present invention as describedby the claims, which follow.

1. A method of attentuating acute kidney injury resulting from a renalinsult in a subject suffering from chronic kidney disease (CKD)comprising administering to the subject a therapeutically effective doseof an oligonucleotide compound which down-regulates expression of atarget gene associated with the acute kidney injury wherein theoligonucleotide compound is administered to the subject in proximity ofthe renal insult, thereby attenuating acute kidney injury in the CKDsubject.
 2. A method of attenuating progression of chronic kidneydisease (CKD) in a subject at risk of CKD progression resulting fromexposure to recurring renal insults comprising administering to thesubject a therapeutically effective dose of an oligonucleotide compoundwhich down-regulates expression of a target gene associated with kidneyinjury wherein the oligonucleotide compound is administered to thesubject in proximity of the renal insult, thereby attenuatingprogression of chronic kidney disease (CKD) in the subject.
 3. A methodof treating or preventing kidney damage in a subject at risk of chronickidney disease (CKD) associated with exposure to a recurrence of renalinsults comprising administering to the subject a therapeuticallyeffective dose of an oligonucleotide compound which down-regulatesexpression of a target gene associated with kidney damage wherein theoligonucleotide compound is administered to the subject in proximity ofeach renal insult, thereby treating or preventing kidney damage in thesubject.
 4. The method according to any one of claims 1-3 wherein theoligonucleotide compound is administered to the subject within about 72hrs pre renal insult to 8 hrs post renal insult.
 5. The method accordingto claim 4 wherein the oligonucleotide compound is administered to thesubject within about 4 hours of the renal insult.
 6. The methodaccording to claim 4 wherein the oligonucleotide compound isadministered to the subject within about 0.5 hours of the renal insult.7. The method according to claim 4 wherein the oligonucleotide isadministered to the subject at about 0-4 hours post the renal insult. 8.The method according to any one of claims 2-3 wherein the renal insultresults in acute kidney injury (AKI).
 9. The method according to any oneof claims 1-3 wherein the renal insult is associated with one or more ofsurgery including cardiovascular surgery; exposure to myoglobinuria,ischemia/reperfusion injury; sepsis; urinary tract obstruction; exposureto a nephrotoxin including a nephrotoxic radiocontrast imaging agent, anantibiotic or a chemotherapeutic agent; proteinuria; increased renalammoniagenesis with interstitial injury; hyperlipidemia;hyperphosphatemia with calcium phosphate deposition.
 10. The methodaccording to claim 9 wherein renal insult is associated withischemia/reperfusion injury or exposure to a nephrotoxin or both. 11.The method according to claim 9 wherein renal insult is associated withischemia/reperfusion ensuing during or following cardiovascular surgeryor cardiopulmonary surgery.
 12. The method according to claim 9 whereinthe renal insult is associated with myoglobinuria.
 13. The methodaccording to claim 9 wherein the renal insult is associated with anephrotoxin.
 14. The method according to claim any one of claims 1-3wherein the target gene associated with kidney injury is a human genewhose expression is up regulated by the renal insult.
 15. The methodaccording to claim 14 wherein the target gene is selected from a genehaving an mRNA sequence shown in Table 1, set forth in any one of SEQ IDNOS: 1-115.
 16. The method according to claim 15 wherein the target geneis selected from p53 and CASP2.
 17. The method according to any one ofclaims 1-3 wherein the oligonucleotide compound is selected from thegroup consisting of an unmodified siRNA, a chemically modified siRNA,shRNA, an aptamer, an antisense molecule, miRNA, and a ribozyme.
 18. Themethod according to claim 17 wherein the oligonucleotide compound ischemically modified siRNA.
 19. The method according to claim 18 whereinthe siRNA has a general double stranded structure: 5′ (N)_(x)-Z 3′(antisense strand) 3′ Z′-(N′)_(y)-z″ 5′ (sense strand) wherein each of Nand N′ is a ribonucleotide which may be unmodified or modified, or anunconventional moiety; wherein each of (N)x and (N′)y is anoligonucleotide in which each consecutive N or N′ is joined to the nextN or N′ by a covalent bond; wherein Z and Z′ may be present or absent,but if present is independently 1-5 consecutive nucleotides ornon-nucleotide moiety covalently attached at the 3′ terminus of thestrand in which it is present; wherein z″ may be present or absent, butif present is a capping moiety covalently attached at the 5′ terminus of(N′)y; each of x and y is independently an integer between 18 and 40;wherein the sequence of (N′)y is substantially complementary to thesequence of (N)x; and wherein (N)x comprises an antisense sequencesubstantially complementary to from about 18 to about 40 consecutiveribonucleotides present in an mRNA of Table 1 set forth in any one ofSEQ ID NO:1-115.
 20. The method according to claim 19 wherein the siRNAis the I5 siRNA compound.
 21. A kit comprising a package containing atherapeutically effective dose of an oligonucleotide compound whichdown-regulates expression of a gene associated with kidney damage in anamount effective to prevent radiocontrast agent induced kidney damage;and a radiocontrast agent in an amount effective to perform aradiographical examination; and optionally instructions for use.
 22. Anoligonucleotide compound which down-regulates expression of a targetgene selected from any one of SEQ ID NO:1-115 for use in therapy forpreventing or attentuating acute kidney injury resulting from renalinsult in a subject suffering from chronic kidney disease (CKD).
 23. Anoligonucleotide compound which down-regulates expression of a targetgene selected from any one of SEQ ID NO: 1-115 for use in therapy forpreventing or attenuating progression of chronic kidney disease (CKD) ina subject at risk of CKD progression resulting from exposure torecurring renal insults.
 24. An oligonucleotide compound whichdown-regulates expression of a target gene selected from any one of SEQID NO:1-115 for use in therapy for treating or preventing kidney damagein a subject at risk of chronic kidney disease (CKD) associated withexposure to a recurrence of renal insults.
 25. Use of oligonucleotidecompound which down-regulates expression of a target gene selected fromany one of SEQ ID NO: 1-115 for preparation of a medicament forpreventing or attentuating acute kidney injury resulting from renalinsult in a subject suffering from chronic kidney disease (CKD).
 26. Useof an oligonucleotide compound which down-regulates expression of atarget gene selected from any one of SEQ ID NO: 1-115 for preparation ofa medicament for preventing or attenuating progression of chronic kidneydisease (CKD) in a subject at risk of CKD progression resulting fromexposure to recurring renal insults.
 27. Use of an oligonucleotidecompound which down regulates expression of a target gene selected fromany one of SEQ ID NO: 1-115 for preparation of a medicament for treatingor preventing kidney damage in a subject at risk of chronic kidneydisease (CKD) associated with exposure to a recurrence of renal insults.28. A composition comprising an oligonucleotide compound whichdown-regulates expression of a target gene selected from any one of SEQID NO:1-115 in an amount effective to attenuate chronic kidney disease,and a carrier.